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Discrimination Effects and Sensitivity Variations in Matrix-Assisted Laser Desorption/ Ionization
Abstract
In matrix-assisted laser desorption/ionization (MALDI) the analyte signal produced depends strongly on the analyte and on the sample preparation procedure. Comparing the results obtained with the dried-drop method with the results obtained with a homogeneous sample preparation procedure, discrimination effects, as well as sensitivity variations, are shown to be dependent on the molecular weight of the analysed compounds. The signal obtained in MALDI is shown to be dependent on the hydrophobicity and basicity of the analyte. With the dried-drop method, in particular, evidence of peripheral sample deposition of hydrophilic compounds is shown. These last effects are due to mass transfer caused by differences in surface activities during solvent evaporation, and are known as Marangoni effects. © 1997 John Wiley & Sons, Ltd.
... As a consequence, these combined factors are thought to compromise the accuracy, precision, and utility [53]. Another challenge in protein determination by MALDI-TOF MS is the reproducibility of peak intensity [54]. The main goal of MALDI protein profiling is to identify differences in peak quality, as well as differences in peak intensities between the investigated sample and the control one. ...
... Unfortunately, the poor reproducibility was found to be one of the main problems during protein analysis by MALDI approach. One of the reasons can be the matrix (co)crystallization-Cohen and Chait [51], as well as Amado et al. [54], have indicated the processes occurring during the sample and matrix preparation. Different matrix molecules may crystallize in different shapes and dimensions; additionally, proteins are likely to accumulate at the droplet periphery, so the composition of the matrix solution and the rate of crystal growth strongly influence the spectral output [53,54]. ...
... One of the reasons can be the matrix (co)crystallization-Cohen and Chait [51], as well as Amado et al. [54], have indicated the processes occurring during the sample and matrix preparation. Different matrix molecules may crystallize in different shapes and dimensions; additionally, proteins are likely to accumulate at the droplet periphery, so the composition of the matrix solution and the rate of crystal growth strongly influence the spectral output [53,54]. Kratzer et al. [55] have described the ion-suppression effect which occurs when an ion suppresses the peak signal of other ions in the sample, and peptides with greater hydrophobicity show the greatest suppression effects. ...
In recent years, matrix-assisted laser desorption/ionization (MALDI) has become the main tool for the study of biological macromolecules, such as protein nano-machines, especially in the determination of their molecular masses, structure, and post-translational modifications. A key role in the classical process of desorption and ionization of the sample is played by a matrix, usually a low-molecular weight weak organic acid. Unfortunately, the interpretation of mass spectra in the mass range of below m/z 500 is difficult, and hence the analysis of low molecular weight compounds in a matrix-assisted system is an analytical challenge. Replacing the classical matrix with nanomaterials, e.g., silver nanoparticles, allows improvement of the selectivity and sensitivity of spectrometric measurement of biologically important small molecules. Nowadays, the nanostructure-assisted laser desorption/ionization (NALDI) approach complements the classic MALDI in the field of modern bioanalytics. In particular, the aim of this work is to review the recent advances in MALDI and NALDI approaches.
... A variety of approaches has been reported to enhance the signal reproducibility of MALDI-TOF MS, either by improving homogeneous matrix crystallization [31,32] or by using an internal standard [33,34] or general calibration procedures [35]. ...
... Finally the peptides were eluted in 50% CH 3 CN/0.1% TFA (30 times up and down). 5 µL of peptides was mixed with 5 µL of matrix HCCA (10 mg/mL in 70% CH 3 CN/0.1% TFA) containing the calibrants, stored at 4°C for 10 min and, in the end, each sample (1.0 µL) was deposited onto three spots of the MALDI target plate kept at 37°C in a dry bath system in order to make constant the evaporation of the solvent, this leading to a more uniform microcrystalline system [31,46] and to a decrease of the Marangoni effect [32]. ...
... The Instrument Control Panel operated in Automatic Control mode (random search pattern in the centre of spot); the following acceptance criteria were set: the intensity mass range was between 30,000 and 60,000; this range allows the instrument to avoid saturation of detector and to obtain a sufficient high signal; molecular mass range was between 800 and 5000; 50 summing spectra, produced an accumulated spectrum with 100 shots/ spectrum (5000 shots); the accumulation of many spectra ameliorates the signal/noise ratio and the differences in ionization rate, intrinsic to this technique [6,12,22]; 5 laser shots pre-scan for search pattern position were run. The initial pre-scan procedure was applied in order to facilitate the fragmentation of big crystals improving the homogeneity of spots; furthermore, pre-scan eliminates dust and some interference substances on the surfaces of spots [32]. We set up our method on 200 attempts and the laser was randomly moved over the centre spots as described by some authors [2] in order to overcome the problems due to non-homogeneous distribution of the analytes and the matrix into the spots. ...
A growing body of literature defines MALDI-TOF MS as a technique for studying plasma and serum, thus enabling the detection of proteins, and the generation of reproducible protein profile mass spectra, potentially able to discriminate correctly different biological systems. In this work, the different steps of the pre-analytical phase that may affect the reproducibility of plasma proteome analysis have been carefully considered. The results showed that the method is highly accurate (9.1%) and precise (8.9%) and the calibration curve for the ACTH (18-39), in human plasma, gave a good correlation coefficient (r>0.99 and r(2)>0.98). The limit of detection (LOD) and the limit of quantification (LOQ), relative intensity, were of 0.5 x 10(-)(9)M and 1.0 x 10(-)(9)M respectively. Thus, an assay has been developed for the detection of low-abundant and low molecular weight proteins, from human plasma, aiming at the identification of new potential biomarkers. The method was tested on plasma from patients with a first diagnosis of pelvic mass. Statistical analysis of plasma profile generated a sub-profile of 17 peptides with their relative abundance able to discriminate patients bearing malignant or benign tumors. The sensitivity and specificity were 85.7% and 80.0% respectively.
... Another phenomenon hindering the use of MALDI for quantitative measurements is the suppression effect, which has also been discussed in a number of publications (19,29,30,32,33,44). Suppression can especially distort the data when complex biological samples are analyzed that contain thousands of components in a wide concentration range. ...
... Recently various factors have been proposed to account for the variability of detected signals (29, 44-49), such as the hydrophobicity and basicity of the peptides, the applied matrix, the amount of various components, or the complexity of the mixture. Several publications claim that peptides that are more hydrophobic or more basic are more detectable (44,50). Promising results were reported when signal reproducibility was improved by using a matrix-comatrix system (21, 48, 51) or using a "sandwich" (52) or other improved sample preparation methodology (1,(53)(54)(55). ...
MALDI-TOF MS has been applied by several groups to relative quantitative measurements. At the same time, the non-quantitative character of this method has been widely reported. We conducted experiments to test the reliability of this technique for quantitation using the statistical method of inverse confidence limit calculation for the first time in this context. The relationship between relative intensities of known amounts of standard peptides and their concentration ratios was investigated. We found that the concentration ratios determined by relative intensity measurements were highly inaccurate and strongly influenced by the molecular milieu of the sample analyzed. Thus, we emphasize the necessity of using the sample itself for calibration. We also performed experiments using an isotope-labeled derivative of the analyte as an internal standard for calibration line generation. As expected, the use of such standard led to a dramatic increase in precision and a less pronounced improvement in accuracy. We recommend performing a similar statistical analysis as a demonstration of reliability for every system where MALDI-TOF MS is used for quantitative measurements.
... However, the use of MALDI-TOF/TOF as an analytical tool in clinical research still needs optimization and evaluation [13]. One of the major limitations of the MALDI-based technique is its relative poor reproducibility in measuring m/z abundances (peak intensity), which may be essential in biomarker discovery where all-or-nothing variations are probably not the most frequent [14]. In addition, LC-MALDI-TOF/TOF analysis generally takes a long time, which is hardly practical when analysing large numbers of samples. ...
... In addition, PCA classified correctly spiked and non-spiked urine aliquots at each spiked peptide concentration (data not shown). Good correlation was found between spiked peptide concentrations and m/z intensity, even though it is known that MALDI ionization yields poor reproducibility of peak intensity, particularly due to poor inter-spot crystallization reproducibility [14] . Quantification precision (CV%: 10–22%) was sufficient here to allow semiquantitative estimation in the low fentomole range. ...
LC-MALDI-TOF/TOF analysis is a potent tool in biomarkers discovery characterized by its high sensitivity and high throughput capacity. However, methods based on MALDI-TOF/TOF for biomarkers discovery still need optimization, in particular to reduce analysis time and to evaluate their reproducibility for peak intensities measurement. The aims of this methodological study were: (i) to optimize and critically evaluate each step of urine biomarker discovery method based on Nano-LC coupled off-line to MALDI-TOF/TOF, taking full advantage of the dual decoupling between Nano-LC, MS and MS/MS to reduce the overall analysis time; (ii) to evaluate the quantitative performance and reproducibility of nano-LC-MALDI analysis in biomarker discovery; and (iii) to evaluate the robustness of biomarkers selection.
A pool of urine sample spiked at increasing concentrations with a mixture of standard peptides was used as a specimen for biological samples with or without biomarkers. Extraction and nano-LC-MS variabilities were estimated by analyzing in triplicates and hexaplicates, respectively. The stability of chromatographic fractions immobilised with MALDI matrix on MALDI plates was evaluated by successive MS acquisitions after different storage times at different temperatures.Low coefficient of variation (CV%: 10-22%) and high correlation (R2 > 0.96) values were obtained for the quantification of the spiked peptides, allowing quantification of these peptides in the low fentomole range, correct group discrimination and selection of "specific" markers using principal component analysis. Excellent peptide integrity and stable signal intensity were found when MALDI plates were stored for periods of up to 2 months at +4 degrees C. This allowed storage of MALDI plates between LC separation and MS acquisition (first decoupling), and between MS and MSMS acquisitions while the selection of inter-group discriminative ions is done (second decoupling). Finally the recording of MSMS spectra to obtain structural information was focused only on discriminative ions in order to minimize analysis time.
Contrary to other classical approaches with direct online coupling of chromatographic separation and on the flight MS and/or MSMS data acquisition for all detected analytes, our dual decoupling strategy allowed us to focus on the most discriminative analytes, giving us more time to acquire more replicates of the same urine samples thus increasing detection sensitivity and mass precision.
... This is based on the fact that the matrix crystallization seldom yields a uniform distribution of the analyte and matrix on the analyzed target, resulting in regions with high and low signal intensity [25]. Thus, in the present experiments we therefore applied a standardized sample preparation protocol, careful calibration, replicated measurements and normalization of the ion intensities to several different matrix peak [24,26,27]. Thereby we achieved quantification when comparing intensities to known concentrations of AF-16 analyzed in the egg yolk medium. ...
Objective: The aim of the present investigation was to determine the level of the active form of the endogenous protein Antisecretory Factor (actAF), and also the active form of AF immunoreactive molecules in the affinity purified egg yolk.
Design: Determination of AF in affinity purified egg yolk by means of in vivo and in vitro methods.
Setting: Sahlgrenska University Hospital and Chalmers University of Technology, both units located in Gothenburg, Sweden. The farm housing the egg-laying hens is situated some 10 miles west to Stockholm.
Samples: Egg yolk collected weekly from hens subjected to AF-stimulating feed for up to 18 weeks.
Methods and main outcome measures: The methods used were:
a/ The in vivo rat ligated jejunal loop assay.
b/ Two variations of ELISA, i.e.
1. An indirect ELISA using a polyclonal antibody against AF-16 antibodies.
2. A competitive enzyme immunoassay for detection of the peptide AF-16 and also related immunoreactive molecules.
c/ Matrix-Assisted Laser Desorption Ionization Mass Spectrometry, (MALDI-MS).
The numeric variables registered represents:
a/ The rat jejunal ligated loop assay demonstrates the influence of AF on the in vivo secretory response (mg/cm) to cholera toxin challenge. The ligated loop is some 12–15 cm long and placed on the mid part of jejunum, and the cholera toxin induced secretion is registered after a 5 h long challenge period.
b/ The indirect ELISA method demonstrates the relative concentration of immunogenic AF peptides/AF-16 peptides and also related immunoreactive compounds by means of absorbance values, while the values of the competitive immunoassay represent the concentration of AF-16 peptides including similar immunoreactive peptides in ng/ml.
c/ The MALDI-MS method provide information about the concentration of the AF-16 peptide down to nanogram per ml. levels after mass spectrometry analysis of the sample.
Results: All methods revealed similar results by demonstrating a continuous increase over time in the collected egg yolk samples. Thus, low AF activity was registered in egg yolk collected in the period of 1–10 weeks of AF-stimulated feeding, significantly higher AF values was registered in yolk collected between 10 and 15 weeks of feeding, while maximal AF concentration was determined after 15 weeks of feeding. Thus, in the period between 15 and 18 weeks of stimulated AF-feeding, no further increase of the endogenous AF activity could be registered despite continuous AF-stimulated feeding.
Conclusion: During the period of AF-stimulated feeding of the egg laying hens the registration of AF concentration in the affinity purified egg yolk samples must be continuously registered over time. The various methods used for determination of AF concentration in the affinity purified egg yolk might all serve as tools in order to achieve the optimal concentration of active AF. Together, these methods will provide information about the optimal AF concentration in the final product consisting of spray dried egg yolk (Salovum®) used for disease treatment.
... The individual features obtained by MALDI-TOF/MS profiling might be handled as categorical variables, but attempts to obtains quantitative data have been broadly used [12][13][14][15], even if MALDI-TOF protein quantification should be based on MS/MS measurement of signal intensities of labelled samples (iTraq or SILAC technologies). In label-free methods, results reliability depends not only on sample preprocessing (preanalytical phase), but also on data acquisition and on data processing (postanalytical phase) [12,[15][16][17][18]. In agreement with this, the intraexperiment reproducibility of MALDI-TOF/MS profiling is reported to vary dramatically for serum/plasma proteins (2-40%) [7,12], and this variation depends on the number and type of the analyzed features. ...
MALDI-TOF profiling of low molecular weight (LMW) peptides (peptidome) usage is limited due to the lack of reproducibility from the confounding inferences of sample preparation, data acquisition and processing. We applied MALDI-TOF analysis to profile urine peptidome with the aims to: 1) compare centrifugal ultrafiltration and dialysis pre-treatments, 2) determine whether using signal LOD (sLOD), together with data normalization, may reduce MALDI-TOF variability. We also investigated the influence of peaks detection on reproducibility. Dialysis allowed to obtain better MALDI-TOF spectra than ultrafiltration. Within the 1000 to 4000 m/z range, we identified 120 and 129 peaks in intra- and inter-assay studies respectively. To estimate the sLOD, serial dilution of pooled urines up to 1/256 were analysed in triplicate. Six data normalization strategies were investigated - the mean, median, internal standard, relative intensity, TIC and linear rescaling normalization. Normalization methods alone performed poorly in reducing features variability while when combined to sLOD adjustment showed an overall reduction in features CVs. Applying a feedback signal processing approach, after median normalization and sLOD adjustment, CVs were reduced from 103% to 26% and 113% to 25% for the intra- and inter-assay respectively, and spectra became more comparable in terms of data dispersion. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... Ion suppression usually occurs when an ion suppresses the signal of another species in the sample. 48 Antibody fragmentation was measured on tissue using a lowest quantity of 41.75 pmol. In this case, three ions (c 20 , c 21 and c 22 ) corresponding to N termini fragments of bevacizumab were measured (Fig. 2C). ...
Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling.
... A detailed description of the ionization mechanisms can be found in specialist reviews for ESI [20] and MALDI [21]. Although both methods allow peptides to be ionized with high sensitivity, there are significant differences between them: first of all, ESI seems to favor the observation of hydrophobic peptides [22,23] whereas MALDI seems to favor the observation of basic and aromatic peptides [22,24,25]; additionally, MALDI seems to discriminate positively Arg-containing peptides, while ESI is less affected by the presence of this amino acid residue [26,27]. This can be an important factor when using proteolytic enzymes other than trypsin [28]. ...
In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry.
... Ions are thought to be formed in this vapour, generally by proton transfer from matrix to analyte (positive ions) or from analyte to matrix (negative ions). In addition, the dried matrix-sample preparation is inhomogeneous, and it has been shown that best ion formation occurs from particular spots in the preparation [43,44]. Although it is known that speci¢c analytes require the use of selected matrices, matrix selection remains empirical. ...
IntroductionScopeReproducibilityWhole Cell MALDI MS of Particular Bacteria Genera and SpeciesStrategies for the Identification of Biomarkers in Whole Cell MALDI MS SpectraConclusions and OutlookReferences
... However, the use of LC-MALDI analysis still needs to be optimized and evaluated [6,7]. To obtain useful information for comparative analysis of samples and differential protein expression using a label-free approach in LC-MALDI techniques, the reproducibility in measuring m/z abundances (peak intensity) and a linear relation between intensity and marker concentration are essential8910. Moreover, although LC-MALDI MS/ MS analysis is a high mass precision technique, it is time consuming, especially if applied to a large number of samples. ...
The use of chromatography coupled with mass spectrometry (MS) analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC) was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC. A chromatographic approach aimed at fractionating the VSMC proteome has never been used before.
This paper describes a strategy for the study of the VSMC proteome. Our approach was based on pre-fractionation with ion exchange chromatography coupled with matrix assisted laser desorption-time of flight mass spectrometry analysis assisted by a liquid chromatography (LC-MALDI-TOF/TOF). Ion exchange chromatography resulted in a good strategy designed to simplify the complexity of the cellular extract and to identify a large number of proteins. Selectivity based on the ion-exchange chemical features was adequate if evaluated on the basis of protein pI. The LC-MALDI approach proved to be highly reproducible and sensitive since we were able to identify up to 815 proteins with a concentration dynamic range of 7 orders of magnitude.
In our opinion, the large number of identified proteins and the promising quantitative reproducibility made this approach a powerful method to analyze complex protein mixtures in a high throughput way and to obtain statistical data for the discovery of key factors involved in VSMC activation and to analyze a label-free differential protein expression.
... 1A shows the MALDI-TOF-MS spectrum acquired in linear mode (1000–12,000 Da) relevant to a healthy donor urine sample simply eluted from a C18 ZipTip ® . As apparent, few peptides were detected in the explored range, likely due to a suppression effect [18,19] on the ionization of low molecular weight (LMW) peptides that could be ascribed to the presence of high molecular weight (HMW) proteins or to limits in the loading capacity of the column. Then, in order to deplete proteins with higher molecular weight and to enrich the lower molecular weight fraction, an ultrafiltration step on 10 or 50 kDa molecular weight cut off membranes was performed before ZipTip ® extraction.Fig. ...
Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples.
Proteomics approaches designed to catalogue all open reading frames (ORFs) under a defined set of growth conditions of an organism have flourished in recent years. However, no proteome has been sequenced completely so far. Here, we generate the largest yeast proteome data set, including 5610 identified proteins, using a strategy based on optimized sample preparation and high-resolution mass spectrometry. Among the 5610 identified proteins, 94.1% are core proteins, which achieves near-complete coverage of the yeast ORFs. Comprehensive analysis of missing proteins showed that proteins are missed mainly due to physical properties. A review of protein abundance shows that our proteome encompasses a uniquely broad dynamic range. Additionally, these values highly correlate with mRNA abundance, implying a high level of accuracy, sensitivity, and precision. We present examples of how the data could be used, including reannotating gene localization, providing expression evidence of pseudogenes. Our near-complete yeast proteome data set will be a useful and important resource for further systematic studies.
Emerging peptide array technologies are able to profile molecular activities within cell lysates. However, the structural diversity of peptides leads to inherent differences in peptide signal to noise ratios (S/N). These complex effects can lead to potentially unrepresentative signal intensities and can bias subsequent analyses. Within mass spectrometry-based peptide technologies, the relation between a peptide’s amino acid sequence and S/N remains largely non-quantitative. To address this challenge, we present a method to quantify and analyze mass spectrometry S/N of two peptide arrays, and we use this analysis to portray quality of data and to design future arrays for SAMDI mass spectrometry. Our study demonstrates that S/N varies significantly across peptides within peptide arrays, and variation in S/N is attributable to differences of single amino acids. We apply supervised machine learning to predict peptide S/N based on amino acid sequence, and identify specific physical properties of the amino acids that govern variation of this metric. We find low peptide-S/N concordance between arrays, demonstrating that different arrays require individual characterization and that global peptide-S/N relationships are difficult to identify. However, with proper peptide sampling, this study illustrates how machine learning can accurately predict the S/N of a peptide in an array, allowing for the efficient design of arrays through selection of high S/N peptides.
Franz Hillenkamp and Michael Karas in the eighties of the last century invented and developed the matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS). Very soon after their invention, MALDI became one of the main tools for the identification of proteins and therefore for proteomics. With MALDI, not only peptides and proteins but also other larger biomolecules like glycans, lipids, and nucleotides are detectable. Thus, it is not surprising that MALDI has become a powerful instrument in medical research and well established in routine medical laboratories. This review highlights the first pioneering years after the invention of MALDI-MS with respect to its impact on applications in medicine. Furthermore, the most important developments of MALDI-based techniques in medical research and routine laboratories are reviewed, such as MALDI mass spectrometric imaging and identification of bacteria.
Quantitative matrix-assisted laser desorption/ionization time of flight (MALDI TOF) approaches have historically suffered from poor accuracy and precision mainly due to the non-uniform distribution of matrix and analyte across the target surface, matrix interferences, and ionization suppression. Tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity as well as improve signal-to-noise ratios by eliminating interferences from chemical noise, alleviating some concerns about dynamic range. However, conventional MALDI TOF/TOF modalities typically only scan for a single MS/MS event per laser shot, and multiplex assays require sequential analyses. We describe here new methodology that allows for multiple TOF/TOF fragmentation events to be performed in a single laser shot. This technology allows the reference of analyte intensity to that of the internal standard in each laser shot, even when the analyte and internal standard are quite disparate in m/z, thereby improving quantification while maintaining chemical specificity and duty cycle. In the quantitative analysis of the drug enalapril in pooled human plasma with ramipril as an internal standard, a greater than 4-fold improvement in relative standard deviation (<10%) was observed as well as improved coefficients of determination (R2) and accuracy (>85% quality controls). Using this approach we have also performed simultaneous quantitative analysis of three drugs (promethazine, enalapril, and verapamil) using deuterated analogues of these drugs as internal standards.
The ideal MALDI/LDI mass spectrometry sample target for an axial TOF instrument possesses a variety of properties. Primarily, it should be chemically inert to the sample, i.e. analyte, matrix and solvents, highly planar across the whole target, without any previous chemical contact and provide a uniform surface to facilitate reproducible measurements without artifacts from previous sample or matrix compounds. This can be hard to achieve with a metal target, which has to be extensively cleaned every time after use. Any cleaning step may leave residues behind, may change the surface properties due to the type of cleaning method used or even cause microscopic scratches over time hence altering matrix crystallization behavior. Alternatively, use of disposable targets avoids these problems. As each possesses the same surface they therefore have the potential to replace the conventional full metal targets so commonly employed. Furthermore, low cost single-use targets with high planarity promise an easier compliance with GLP guidelines as they alleviate the problem of low reproducibility due to inconsistent sample/matrix crystallization and changes to the target surface properties. In our tests, polymeric metal nano-coated targets were compared to a stainless steel reference. The polymeric metal nano-coated targets exhibited all the performance characteristics for a MALDI MS sample support, and even surpassed the - in our lab commonly used - reference in some aspects like limit of detection. The target exhibits all necessary features such as electrical conductivity, vacuum, laser and solvent compatibility.
In the last decade matrix-assisted laser desorption/ionization (MALDI) has become a powerful tool in biological mass spectrometry (MS). At present, electrospray ionization (ESI) and MALDI are the two major soft ionization techniques for the intact analysis of macromolecules by mass spectrometry. While MALDI-MS provides inherently fast data acquisition and spectra facile for data analysis, ESI is considered to be the softer of the two techniques and is therefore the method of choice for the analysis of labile substituents of posttranslationally modified peptides and proteins, as well as studies of non-covalent complexes.
The two most common methods used to place charged high mass molecules into the gas phase, electrospray ionization and matrix-assisted laser desorption/ionization (maldi), are described. Because of its dominant use with synthetic polymers, the maldi method is described in detail, including sample preparation and data analysis and the problems this method poses. The time-of-flight mass spectrometry system is also described in detail since it is the most common in use today for synthetic polymers. A variety of applications of maldi to synthetic polymers is reviewed. This includes molecular mass distribution, molecular composition distribution of copolymers, polymer architecture, combinations of maldi-tof-ms with size exclusion chromatography and other forms of polymer chromatography, and the use of maldi in elucidating polymer chemical reaction mechanisms.
Mass spectrometry (MS)-based analysis using enzymatic digestion is widely used for protein sequencing and characterization. The large number of peptides generated from proteolysis, however, suppresses the signal of peptides with low ionization efficiency, thus precluding their observation and analysis. This study describes a technique for improved analysis of peptic peptides by adding the synthetic receptor cucurbit[7]uril (CB[7]), which binds selectively to peptides with N-terminal aromatic residues. Capturing the N-terminal phenylalanine (Phe) of peptides using CB[7] enhances the peptide abundances both in electrospray ionization MS and in matrix-assisted laser desorption ionization MS. Moreover, collision-induced dissociation (CID) of the CB[7]•peptide complex ions generates b- and y-type fragment ions with higher sequence coverage than those generated with uncomplexed peptides. The signal enhancement mediated by CB[7] is attributed to an increase in the peptide proton affinities upon CB[7] complexation. The mechanistic details of the fragmentation process are discussed based on the structures of the complex ions obtained from ion mobility (IM) measurements and molecular modeling. This study demonstrates a novel and powerful approach to the enhancement of protein and peptide analysis using a synthetic receptor, without the need for new instrumentation, chemical modifications, or specialized sample preparation. The simplicity and potential generality of this technique should provide a valuable asset in the toolbox of routine protein and peptide analysis.
This chapter seeks to provide a brief overview of the present capabilities and the future possibilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) using cluster primary ions in the study of biological cells and tissue. There are basically two types of ToF-SIMS instrumentation available at present. By far, the majority of instruments utilize a pulsed primary beam and a reflectron time of flight mass spectrometer. Very recently a new concept of instrument has appeared that uses a DC primary beam and a hybrid two-stage mass spectrometer arrangement. First, the chapter summarizes the benefits common to both instruments and then outlines those specific to the new ToF-SIMS platforms. The challenges facing ToF-SIMS in biological studies is then discussed, followed by some illustrative examples of the present status of ToF-SIMS in biological analyses.
Over the past decade, mass spectrometry (MS) has been increasingly employed in structural analyses of glycan structures in glycoproteins. Electrospray ionization (ESI)-MS is particularly useful for this purpose. A number of reports on the direct MS analysis of digested glycopeptides have appeared recently. However, the use of MS-based techniques for the complete glycostructure of glycopeptides remains a challenging task. In this study, we report on a method for determining glycostructures in glycopeptides originating from the b2-glycoprotein I (b2-GPI). The method involves the use of liquid chromatography (LC)-ESI MS. While MS/MS spectra can provide useful information concerning glycostructures, additional MS information is needed to completely determine the composition and sequence of a glycan. This complicates the analysis, because the MS/MS data must be augmented by data from the corresponding MS spectrum involving significant in-source fragmentation of MS spectra in LC-ESI MS. In LC quadrupole-time-of-flight (Q-TOF) MS, triantennary and biantennary glycans of b2-GPI were mainly determined by combining MS and MS/MS spectral data. Finally, LC-Q-TOF MS was used to determine 24 kinds of glycopeptides at four N-glycosylation sites in b2-GPI. It also turned out that LC-Q-TOF MS was useful for the detection of glycans attached to hydrophobic peptides and other peptides that were undetectable by matrix-assisted laser desorption/ionization (MALDI)-Q-TOF MS.
Background:
Gingival crevicular fluid (GCF) may be a source of new biomarkers of periodontitis/gingivitis. However, the minute volumes of GCF harvested in healthy sites are a serious drawback. We evaluated how pre-analytical and analytical variables concerning GCF collection and processing, could significantly influence quality and reproducibility of MALDI-TOF profiles.
Methods:
GCF was collected from healthy sites. The use of paper strips vs paper points was compared. Peptides and proteins were extracted by centrifugal elution in different solutions, at different accelerations, with and without protease inhibitor cocktail (PIC). Finally, we evaluated how matrix composition and matrix/sample volume ratio affect the reproducibility of MALDI-TOF profiles.
Results:
Trifluoroacetic acid elution generated richer gingival fingerprints compared to acetic acid, independently of the collection device. Centrifugation speed and PIC supplementation did not change GCF profiles. A fine modulation of matrix composition and matrix/sample volume ratio resulted in a satisfactory reproducibility (CV less than 10% for peak area and signal-to-noise ratio).
Conclusion:
An optimized procedure, enabling generation of reproducible MALDI-TOF profiles from limited volume of GCF, is proposed. These fingerprints may serve as reference for future studies oriented to the maintenance and preservation of good gingival status and to discovery biomarkers of periodontitis/gingivitis.
Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution.
Human urine is an attractive and informative biofluid for medical diagnosis, which has been shown to reflect the (patho)-physiology of not only the urogenital system, but also others such as the cardiovascular system. For this reason, many studies have concentrated on the study of the urine proteome, aiming to find relevant biomarkers that could be applied in a clinical setting. However, this goal can only be achieved after reliable quantitative and qualitative analysis of the urinary proteome. In the last two decades, MS-based platforms have evolved to become indispensable tools for biomarker research. In this review, we will present and compare two of the most clinically relevant analytical platforms that have been used for the study of the urinary proteome, namely CE-based ESI-MS and classical MALDI-MS. These platforms, although not directly comparable, have been extensively used in proteomic profiling and therefore their comparison is fundamentally relevant to this field.
Polyamidoamine (PAMAM) are synthetic dendrimers which present attractive properties for the biological and biomedical fields, as they proved to be efficient drug and gene carriers. In order to increase their transfection efficiency, chemical modifications of the amino end-groups had been reported. In this work, the synthesis of the ammonia-cored G1(N) PAMAM and the consecutive chemical modification with glycine or phenylalanine amino-acids were monitored using the coupling of thin layer chromatography (TLC) with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Thus, the monitoring of the PAMAM synthesis included the identification of the by-products such as defective structures of PAMAM dendrimers as well as the study of phenylalanine-grafted PAMAM oligomer distribution.
A standard dried-droplet preparation using 2,5-dihydroxybenzoic acid (2,5-DHBA) as the matrix results in a large variation in signal intensity and poor shot-to-shot reproducibility in matrix-assisted laser desorption/ionization (MALDI). We expected that the differences can be attributed to the nature of the crystal structures in the region of the "sweet spot" within the MALDI samples. 2,5-DHBA crystals with and without analytes on a target plate obtained by means of a dried-droplet preparation contain two polymorphs, which can be distinguished by Raman spectra. In comparing the Raman image with the MS image, a clear correlation between the signal distribution of glycopeptides and hydrophilic peptides and the specific crystal form of 2,5-DHBA could be made. The ionization of hydrophobic peptides appears to proceed in both types of polymorphic crystals. In addition, the derivatization of glycopeptides with a pyrene group enabled us to detect glycopeptides regardless the crystal form. As the result, the number of sweet spots increased and MS spectra with a high signal intensity were obtained. The results suggest that the introduction of a hydrophobic/aromatic moiety to glycopeptides results in a more successful MALDI analysis due to the effective incorporation of the analyte into matrix crystals.
The aim of this study was to investigate the quality and reproducibility of mass spectra derived from a matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) platform in a patient population undergoing carotid endarterectomy. Plasma samples were either digested with trypsin or left undigested, fractionated with either C18 or weak cation exchange (WCX) columns and analysed by MALDI-TOF MS. Quality of mass spectra for each method was assessed by baseline correction (lower area under the curve ratio indicating higher quality) and signal-to-noise ratio. Mean coefficient of variation (CV%) assessed reproducibility between repeated experiments and methods. Identified mass peak intensity differences were assessed for consistency across repeated experiments. Plasma from six patients was analysed. The quality of mass spectra was significantly better when derived from digested plasma fractionated by either WCX or C18 methods compared to undigested plasma fractionated by WCX (analysis of variance, p < 0.05). Mean CV% for repeated experiments was 18% and 28% for WCX and C18 fractionated digested plasma, respectively. A small number of differences in mass peak intensities were consistently observed in repeated experiments. Repeated experiments are required to confidently identify non-random mass peak intensity differences as putative plasma biomarkers that merit further investigation.
Despite the progress in performance of MS-driven analytical methods during the last decade with regard to sensitivity and selectivity, the identification of phosphorylation sites is still not a trivial task. Thus, today no single method can reliably detect and characterize all modified residues in a phosphoprotein and far most successful analysis strategies for phosphoprotein and phosphoproteome, including multiple levels of enrichment and separation methods as well as biological follow-up analysis. However, recent improvements in MS have spawned improved and far more robust analytical strategies. An improved effiency of enrichment and separation techniqes on both peptide and protein level, the improved data quality by ECD or ETD peptide fragmentation and the improved confidence in phosphopeptide detection by MS 3 phosphopeptide sequencing using high mass accuracy FTICR-based mass spectrometers have enabled multiple comprehensive studies of protein phsophorylation. Last, but not least, multiple complementary MS-driven strategies for relative and absolute quantitation of protein phosphorylation will ease rapid investigations of signal transduction systems and, thus, provide the basis for great advances.
The fast and univocal identification of different species in mixtures of pollen grains is still a challenge. Apart from microscopic evaluation and Raman spectroscopy, no other techniques are available.
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was applied to the analysis of extracts of single pollen grains and pollen mixtures. Pollen grains were fixed, treated and covered with matrix directly on the MALDI target.
Clearly resolved MALDI ion intensity images could be obtained enabling the identification of single pollen grains in a mixture.
Our results demonstrate the potential and the suitability of MALDI imaging mass spectrometry as an additional method for the identification of pollen mixtures. Copyright
An emerging application of matrix-assisted laser desorption ionization (MALDI) mass spectrometry is the analysis of low molecular weight (LMW) compounds, often via coupled liquid chromatography - MALDI-MS methods. However, in many cases, the low molecular weight region of MALDI mass spectra is obscured by the presence of signals originating from the matrix, suggesting that the development of tethered MALDI matrixes may be required to optimize MS performance for such compounds. To gain insight into potential sites for covalent attachment of MALDI matrixes, we have systematically investigated the role played by a variety of functional group motifs in determining matrix efficiency for three common MALDI matrixes, as judged both by total signal intensity and background noise from matrix decomposition for a set of LMW compounds. A series of allyl derivatives of standard matrixes was prepared, and the efficiency of these materials in the MALDI experiment was measured. All modifications of established matrixes, e.g., 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxycinnamic acid (CHCA), and caffeic acid (CA), or close analogues led to decreased absolute signal intensity and signal-to-background levels. Improved performance was generally observed with (i) the presence of a phenolic group (carboxylic acids were less effective) (ii) crystalline derivatives, and (iii) compounds that had high extinction coefficients at wavelengths near to that of the exciting laser (337 nm). The most interesting derivatives were the O-allyl ether (15) and N-allyl amide (16) of caffeic acid. These compounds did not facilitate signals from all four analytes tested. However, the observed spectra contained fewer signals from the matrix than from the parent compound CA. These compounds demonstrate that functionalization of MALDI matrixes, ultimately leading to tethered matrixes, is possible without jeopardizing signal intensity.
Matrix assisted inlet ionization (MAII) is a method in which a matrix:analyte mixture produces mass spectra nearly identical to electrospray ionization without the application of a voltage or the use of a laser as is required in laserspray ionization (LSI), a subset of MAII. In MAII, the sample is introduced by, for example, tapping particles of dried matrix:analyte into the inlet of the mass spectrometer and, therefore, permits the study of conditions pertinent to the formation of multiply charged ions without the need of absorption at a laser wavelength. Crucial for the production of highly charged ions are desolvation conditions to remove matrix molecules from charged matrix:analyte clusters. Important factors affecting desolvation include heat, vacuum, collisions with gases and surfaces, and even radio frequency fields. Other parameters affecting multiply charged ion production is sample preparation, including pH and solvent composition. Here, findings from over 100 compounds found to produce multiply charged analyte ions using MAII with the inlet tube set at 450 °C are presented. Of the compounds tested, many have -OH or -NH(2) functionality, but several have neither (e.g., anthracene), nor aromaticity or conjugation. Binary matrices are shown to be applicable for LSI and solvent-free sample preparation can be applied to solubility restricted compounds, and matrix compounds too volatile to allow drying from common solvents. Our findings suggest that the physical properties of the matrix such as its morphology after evaporation of the solvent, its propensity to evaporate/sublime, and its acidity are more important than its structure and functional groups.
Human serum has been widely employed clinically for diagnosing various fatal diseases. However, the concentration of most proteins in human serum is too low to be directly measured using normal analytical methods. In order to obtain reliable analytical results from proteomic analysis of human serum, appropriate sample preparation is essential. A combined off-line analytical technique of gel chromatography and matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been successfully established to separate proteins for MS analysis. Using these combined techniques, 176 mass signal peaks of proteins/peptides were found in 6 of 18 fractions from normal male serum (NMS) in the presence of buffer consisting of NH4HCO3 and acetonitrile. A simple gel chromatography column packed with Sephadex G-50 removed most signal-suppressing compounds such as salts and high abundance proteins (HAP). The molecular mass to charge (m/z) ratios of differential peptides revealed in serum of male patient with liver-cancer (LCMPS) compared to NMS were 5365, 5644 and 6462, and these peptides can be used as biomarkers to clinically diagnose liver-cancer. The simple and convenient chromatographic method described here is not only superior to recently described HPLC separation for MS analysis, but also reveals many novel and significant serum biomarkers for the clinical diagnosis of various diseases.
Microscale cleaning procedures for peptides, proteins and glycoproteins prior to matrix-assisted laser desorption ionization (MALDI) mass spectrometric (MS) analysis, based on different mechanisms – drop dialysis, ultrafiltration, reverse phase adsorption and gelfiltration, are being compared. The selected standard (glyco) proteins cover the mass range of 5000–70 000 Da. Individual components and mixtures containing up to five components have been used for the investigation. Five different procedures were applied and evaluated with regard to a subsequent positive ion MALDI time-of-flight MS analysis, which requires samples with relative low salt/buffer content. All methods were adapted for protein samples in the low picomole range. Drop dialysis, ultrafiltration, reverse phase adsorption in two different forms and gelfiltration are shown to be useful for proteins and partially for peptides and glycoproteins. It turned out that glycoproteins are more difficult to handle, due to the relatively high sample losses during the desalting procedures. Gelfiltration and reverse phase adsorption are the least time consuming methods, whereas drop dialysis and ultrafiltration are well suited for working on many samples in parallel. However, reverse phase adsorption and ultrafiltration offer the highest enrichment factors. For proteins available only in large sample volumes gelfiltration and reverse phase adsorption are the methods of choice.
Off-line coupling of capillary IEF (CIEF) with matrix-assisted laser desorption/ionization mass spectrometry was utilized for the analysis of human blood serum. Serum proteins were initially separated by CIEF, and fractions of the isoelectric separation were eluted sequentially to a MALDI-TOF MS sample target. During pressure elution of the CIEF sample, voltage was maintained across the capillary system utilizing a sheath flow arrangement to minimize band broadening induced by the laminar flow field. Both pI and mass information were obtained from the complex biological sample, similar to traditional 2-DE techniques, and the platform was faster (hours versus days), more automatable, and simpler than 2-DE. The volume of raw sample present in the actual analysis was approximately 100 nL, making this technique well suited for very rare specimens. Additionally, the speed and simplicity of the technology make it an attractive technique for performing initial comparative analyses of complex samples.
Recent developments in desorption/ionisation mass spectrometry techniques have made their application to biological analysis a realistic and successful proposition. Developments in primary ion source technology, mainly through the advent of polyatomic ion beams, have meant that the technique of secondary ion mass spectrometry (SIMS) can now access the depths of information required to allow biological imaging to be a viable option.Here the role of the primary ion C60+ is assessed with regard to molecular imaging of lipids and pharmaceuticals within tissue sections. High secondary ion yields and low surface damage accumulation are demonstrated on both model and real biological samples, indicating the high secondary ion efficiency afforded to the analyst by this primary ion when compared to other cluster ion beams used in imaging. The newly developed 40 keV C60+ ion source allows the beam to be focused such that high resolution imaging is demonstrated on a tissue sample, and the greater yields allow the molecular signal from the drug raclopride to be imaged within tissue section following in vivo dosing.The localisation shown for this drug alludes to issues regarding the chemical environment affecting the ionisation probability of the molecule; the importance of this effect is demonstrated with model systems and the possibility of using laser post-ionisation as a method for reducing this consequence of bio-sample complexity is demonstrated and discussed.
Since the early days of matrix-assisted laser desorption/ionization (MALDI), the definition of a unified theory on the MALDI process is a major challenge. The new results presented in this paper clearly show that the idea of a uniform MALDI mechanism that accounts for the spreading applications has to be given up. Based on different preparation protocols distinct differences in the desorption/ionization process for carbohydrates in contrast to peptides/proteins are elucidated. Although isolated/incorporated and cluster-desorbed peptides/proteins are effectively entrained and cooled within the expanding plume of matrix clusters, as shown by a low degree of metastable analyte-ion fragmentation, a laser desorption and gas-phase cationization mechanism can be confirmed as the dominant part in ionization for neutral oligosaccharides which can be initiated even for particulate analyte material or deposits onto a matrix surface. The previously presented “lucky survivor-model” on cluster desorption of preformed ions thus needs this extension.
We describe a novel approach to infrared matrix-assisted laser desorption-ionization mass spectrometry using a tunable, picosecond pulse laser to selectively excite specific modes of a solid, thereby creating a high local density of vibrational quanta. The concept is based on recent results from our experiments employing a free-electron laser to explore ‘matrix-less’ mass spectrometry in which an infrared chromophore intrinsic to the sample, rather than an exogenous matrix, is excited by the laser. Examples from both environmental mass spectrometry and a proteomics-driven research project are presented, showing how the principle of selective vibrational excitation can be used to make possible novel and useful ion generation protocols. We conclude with an analysis of possible mechanisms for the phenomena of infrared desorption, ablation and ionization using very short laser pulses. Prospects for achieving similar results with more conventional laser sources are discussed.
Protein profiling with high-throughput sample preparation and MALDI-TOF MS analysis is a new potential tool for diagnosis of human diseases. However, analytical reproducibility is a significant challenge in MALDI protein profiling. This minireview summarizes studies of reproducibility of MALDI protein profiling and current approaches to improve its analytical performance.
The PubMed database was searched using combinations of the following search terms: MALDI, SELDI, reproducibility, variation, precision, peak intensity, quantification, peptide, biomarkers, and proteomics. Acceptance criteria were detailed reports on the reproducibility with MALDI protein profiling and studies describing efforts to improve the analytical performance with this technology.
The reported intraexperiment CVs of the peak intensity vary highly between individual protein peaks, with the reported mean CV of the peak intensity varying among studies from 4% to 26%. There is additional interexperiment variation in peak intensity. Current approaches to improve the analytical performance of MALDI protein profiling include automated sample processing, extensive prefractionation strategies, immunocapture, prestructured target surfaces, standardized matrix (co)crystallization, improved MALDI-TOF MS instrument components, internal standard peptides, quality-control samples, replicate measurements, and algorithms for normalization and peak detection.
Further evaluation and optimization of MALDI-TOF MS is recommended before use in routine analysis.
The possibility of exploiting the analytical power of mass spectrometry to image the chemistry of biological and similarly complex materials without the use of tags and with good spatial resolution is seductive. The status, strengths, weaknesses and complementarity of the three main techniques are briefly reviewed and assessed.
Enzymatic synthesis of oligopeptides from l-phenylalanine ethyl ester hydrochloride (l-Phe-Et.HCl) and other l-form hydrophobic amino acid ester hydrochlorides in water miscible organic cosolvents was studied. Different proteases, water miscible cosolvents, and effect of different ratios of water miscible cosolvents for protease-catalyzed oligo-phenylalanine [oligo(l-Phe)] were compared. The importance of the use of water miscible cosolvents in transforming reactions from heterogeneous to homogeneous conditions as a potent medium engineering tool for protease-catalyzed oligopeptide synthesis is highlighted. For example, at 0.125 M l-Phe-Et.HCl, 20% (v/v) methanol, 18.6 mg/mL bromelain, in phosphate buffer (0.25M, pH 8), 40 degrees C, for 3 h, oligo(l-Phe) precipitated from the solution to yield 45 +/- 5%, in contrast, in the absence of cosolvent oligo(l-Phe) yield of 29 +/- 5% was obtained. The following reaction conditions were optimized for bromelain catalyzed oligo(l-Phe) synthesis: pH, temperature, substrate, enzyme, and cosolvent concentrations. DP(avg) and chain length distribution in the product peptides were investigated by (1)H NMR and MALDI-TOF.
In principle mass spectral imaging has enormous potential for discovery applications in biology. The chemical specificity of mass spectrometry combined with spatial analysis capabilities of liquid metal cluster beams and the high yields of polyatomic ion beams should present unprecedented ability to spatially locate molecular chemistry in the 100 nm range. However, although metal cluster ion beams have greatly increased yields in the m/z range up to 1000, they still have to be operated under the static limit and even in most favorable cases maximum yields for molecular species from 1 µm pixels are frequently below 20 counts. However, some very impressive molecular imaging analysis has been accomplished under these conditions. Nevertheless although molecular ions of lipids have been detected and correlation with biology is obtained, signal levels are such that lateral resolution must be sacrificed to provide a sufficient signal to image. To obtain useful spatial resolution detection below 1 µm is almost impossible. Too few ions are generated! The review shows that the application of polyatomic primary ions with their low damage cross-sections offers hope of a new approach to molecular SIMS imaging by accessing voxels rather than pixels to thereby increase the dynamic signal range in 2D imaging and to extend the analysis to depth profiling and 3D imaging. Recent data on cells and tissue analysis suggest that there is, in consequence, the prospect that a wider chemistry might be accessible within a sub-micron area and as a function of depth. However, these advances are compromised by the pulsed nature of current ToF-SIMS instruments. The duty cycle is very low and results in excessive analysis times, and maximum mass resolution is incompatible with maximum spatial resolution. New instrumental directions are described that enable a dc primary beam to be used that promises to be able to take full advantage of all the capabilities of the polyatomic ion beam. Some new data are presented that suggest that the aspirations for these new instruments will be realized. However, although prospects are good, the review highlights the continuing challenges presented by the low ionization efficiency and the complications that arise from matrix effects.
Detection limits of poly(ethylene glycol) were examined in the mass range 2000-6000 Da. Using an aerospray sample deposition technique, highly uniform sample surfaces were produced. This method allows signal averaging of spectra from up to 400 shots on the same sample spot. It is found that, as the material available for desorption is decreased, the overall average sample consumption per shot is decreased. Experimentally determined detection limits of 40 and 280 fmol (based on the average molecular masses of 2000 and 6000) were found for PEG 2000 and PEG 6000, respectively. The sample spectra show oligomer distributions in agreement with their higher concentration counterparts. However, at the lowest signal-to-noise levels, oligomers at the extremes of the distribution are no longer detected, making the polymer distribution appear to be narrower in mass range.
Matrix-assisted laser desorption/ionization time-of-flight mass spectra of proteins in cerebrospinal fluid analyzed without prior purification are presented. Less than 100 fmol amounts of proteins in the 10,000 to 20,000 u mass range and linked to human disease (multiple sclerosis, Alzheimer's disease, and stroke) were detected in a complex mixture of proteins and peptides, in the presence of high concentrations of salts, lipids and free amino acids. The mass resolution was sufficient to distinguish between the non-hydroxylated and hydroxylated forms of a 13,400 u protein. Simple fractionation of the cerebrospinal fluid using microbore-reversed phase high performance liquid chromatography improved signal-to-noise ratios in the mass spectra. High-accuracy peptide mass mapping and database searching were utilized to confirm the identity of several proteins. The presented results show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry could be used as a tool to perform rapid screening of chemically altered proteins in small volumes of biological fluids.
The techniques of enzymatic and chemical peptide ladder sequencing, coupled with ultraviolet - matrix assisted laser desorption/ionization - mass spectrometry (UV-MALDI-MS) have been improving continuously in the last five years and have now become important tools for primary structure identification. In this work, signal suppression effects, appearing in UV-MALDI-MS (excitation 337 nm) of ladder peptides, were investigated using the 17-amino acid peptide dynorphin A. We show, with examples of simple "two-peptide" systems and more complex "multi-peptide" systems, that suppression effects do in fact exist. The magnitude of the observed suppression is strongly dependent upon both the nature and the amount of the suppressing peptide. Suppression behavior of individual ladder peptides was investigated on equimolar mixtures of ten ladder peptides. Significant signal suppression was recorded for all ladder peptides, with some of them being approximately 170 times lower in signal intensity than the pure, i.e., unsuppressed peptide at the same concentration. For the investigated system--dynorphin A, 4-hydroxy-alpha-cyanocinnamic acid (4-HCCA) matrix, UV excitation--a correlation between the extent of suppression and an intractable combination of peptide hydrophobicity and the presence of several basic amino acids can be seen.
Ribosomes from the K-12 strain of Escherichia coli were analyzed with good sensitivity and high mass accuracy using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fifty-five of the 56 subunit proteins were observable. Mass spectral peak locations were consistent with previously reported post-translational modifications involving N-terminal methionine loss, methylation, thiomethylation, and acetylation for all but one case. The speed and accuracy of mass spectrometry make it a good candidate for phylogenetic studies of ribosomes and the observation of posttranslational modifications in other organisms.
This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples.
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