A study of reproducibility of guanidination-dimethylation labeling and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry for relative proteome quantification

The combination of dimethylation after guanidination (2MEGA) isotope labeling with microbore liquid chromatography (LC)-matrix-assisted laser desorption ionization (MALDI) MS and MS/MS [C. Ji, N. Guo, L. Li, J. Proteome Res. 4 (2005) 2099] has been reported as a promising strategy for abundance ratio-dependent quantitative proteome analysis. A critical step in using this integrated strategy is to set up the abundance ratio threshold of peptide pairs, above which the peptide pairs are used for quantifying and identifying the protein that is considered to be differentially expressed between two different samples. The threshold is determined by technical variation (i.e., the overall abundance ratio variation caused by the experimental process including sample workup, MS analysis and data processing) as well as biological variation (i.e., the abundance ratio variation caused by the biological process including cell growth), which can be defined and assessed by a coefficient of variation (CV). We have designed experiments and measured three different levels of variations, starting with the same membrane protein preparation, the same batch of cells and three batches of cells from the same cell line grown under the same conditions, respectively. It is shown that technical variation from the experimental processes involved in 2MEGA labeling LC-MALDI MS has a CV of <15%. In addition, the measured biological variation from cell growth was much smaller than the measured technical variation. From the studies of the occurrence rate of outliers in the distribution of the abundance ratio data within a comparative dataset of peptide pairs, it is concluded that, to compare the proteome changes between two sets of cultured cells without the use of replicate experiments, a relative abundance ratio of greater than 2X or less than 0.5X (X is the average abundance ratio of the dataset) on peptide pairs can be used as a stringent threshold to quantify and identify differentially expressed proteins with high confidence.     

Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis

Isotope labeling liquid chromatography–mass spectrometry (LC–MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.     

Prerequisites for peptidomic analysis of blood samples: II. Analysis of human plasma after oral glucose challenge -- a proof of concept

Mass spectrometric plasma analysis for biomarker discovery has become an exploratory focus in proteomic research: the challenges of analyzing plasma samples by mass spectrometry have become apparent not only since the human proteome organization (HUPO) has put much emphasis on the human plasma proteome. This work demonstrates fundamental proteomic research to reveal sensitivity and quantification capabilities of our Peptidomics technologies by detecting distinct changes in plasma peptide composition in samples after challenging healthy volunteers with orally administered glucose. Differential Peptide Display (DPD) is a technique for peptidomics studies to compare peptides from distinct biological samples. Mass spectrometry (MS) is used as a qualitative and quantitative analysis tool without previous trypsin digestion or labeling of the samples. Circulating peptides (< 15 kDa) were extracted from 1.3 mL plasma samples and the extracts separated by liquid chromatography into 96 fractions. Each fraction was subjected to MALDI MS, and mass spectra of all fractions were combined resulting in a 2D-display of > 2,000 peptides from each sample. Endogenous peptides that responded to oral glucose challenge were detected by DPD of pre-and post-challenge plasma samples from 16 healthy volunteers and subsequently identified by nESI-qTOF MS. Two of the 15 MS peaks that were significantly modulated by glucose challenge were subsequently identified as insulin and C-peptide. These results were validated by using immunoassays for insulin and C-peptide. This paper serves as a proof of principle for proteomic biomarker discovery down to the pM concentration range by using small amounts of human plasma.     

imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation

An integrated sample preparation method, termed “imFASP”, which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50–500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples.     

Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: tandem mass spectrometry comparison using MALDI-TOF,ESI-QTOF,and ESI-ion trap mass spectrometers

Quantitative or comparative proteome analysis was initially performed with 2-dimensional gel electrophoresis with the inherent disadvantages of being biased towards certain proteins and being labor intensive. Alternative mass spectrometry-based approaches in conjunction with gel-free protein/peptide separation have been developed in recent years using various stable isotope labeling techniques. Common to all these techniques is the incorporation, biosynthetically or chemically, of a labeling moiety having either a natural isotope distribution of hydrogen, carbon, oxygen, or nitrogen (light form) or being enriched with heavy isotopes like deuterium, (13)C, (18)O, or (15)N, respectively. By mixing equal amounts of a control sample possessing for instance the light form of the label with a heavy-labeled case sample, differentially labeled peptides are detected by mass spectrometric methods and their intensities serve as a means for direct relative protein quantification. While each of the different labeling methods has its advantages and disadvantages, the endoprotease (16)O-to-(18)O catalyzed oxygen exchange at the C-terminal carboxylic acid is extremely promising because of the specificity assured by the enzymatic reaction and the labeling of essentially every protease-derived peptide. We show here that this methodology is applicable to complex biological samples such as a subfraction of human plasma. Furthermore, despite the relatively small mass difference of 4 Da between the two labeled forms, corresponding to the exchange of two oxygen atoms by two (18)O isotopes, it is possible to quantify differentially labeled proteins on an ion trap mass spectrometer with a mass resolution of about 2000 in automated data dependent LC-MS/MS acquisition mode. Post column sample deposition on a MALDI target parallel to on-line ESI-MS/MS enables the analysis of the same compounds by means of ESI- and MALDI-MS/MS. This has the potential to increase the confidence in the quantification results as well as to increase the sequence coverage of potentially interesting proteins by complementary peptide ionization techniques. Additionally the paired y-ion signals in tandem mass spectra of (16)O/(18)O-labeled peptide pairs provide a means to confirm automatic protein identification results or even to assist de novo sequencing of yet unknown proteins.     

Combining capillary electrophoresis matrix-assisted laser desorption/ionization mass spectrometry and stable isotopic labeling techniques for comparative crustacean peptidomics

Herein we describe a sensitive and straightforward off-line capillary electrophoresis (CE) matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) interface in conjunction with stable isotopic labeling (SIL) technique for comparative neuropeptidomic analysis in crustacean model organisms. Two SIL schemes, including a binary H/D formaldehyde labeling technique and novel, laboratory-developed multiplexed dimethylated leucine-based isobaric tagging reagents, have been evaluated in these proof-of-concept experiments. We employ these isotopic labeling techniques in conjunction with CE-MALDI-MS for quantitative peptidomic analyses of the pericardial organs isolated from two crustacean species, the European green crab Carcinus maenas and the blue crab Callinectes sapidus. Isotopically labeled peptide pairs are found to co-migrate in CE fractions and quantitative changes in relative abundances of peptide pairs are obtained by comparing peak intensities of respective peptide pairs. Several neuropeptide families exhibit changes in response to salinity stress, suggesting potential physiological functions of these signaling peptides.     

Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z? > ?2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

Protein- versus peptide fractionation in the first dimension of two-dimensional high-performance liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry for qualitative proteome analysis of tissue samples

The availability of robust and highly efficient separation methods represents a major requirement for proteome analysis. This study investigated the characteristics of two different gel-free proteomic approaches to the fractionation of proteolytic peptides and intact proteins, respectively, in a first separation dimension. Separation and mass spectrometric detection by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) were performed at the peptide level in both methods. Bottom-up analysis (BU) was carried out employing well established peptide fractionation in the first separation dimension by strong cation-exchange chromatography (SCX), followed by ion-pair reversed-phase chromatography (IP-RPC) in the second dimension. In the semi-top-down approach (STD), which involved intact protein fractionation in the first dimension, the separation mode in both dimensions was IP-RPC utilizing monolithic columns. Application of the two approaches to the proteome analysis of proteins extracted from a tumor tissue revealed that the BU method identified more proteins (1245 in BU versus 920 in STD) while STD analysis offered higher sequence coverage (14.8% in BU versus 17.5% in STD on average). The identification of more basic and larger proteins was slightly favored in the BU approach, most probably due to higher losses of these proteins during intact protein handling and separation in the STD method. A significant degree of complementarity was revealed by an approximately 33% overlap between one BU and STD replicate, while 33% each of the protein identifications were unique to both methods. In the STD method, peptides obtained upon digestion of the proteins contained in fractions of the first separation dimension covered a broad elution window in the second-dimension separation, which demonstrates a high degree of “pseudo-orthogonality” of protein and peptide separation by IP-RPC in both separation dimensions.     

Evaluation of isotope-coded protein labeling (ICPL) in the quantitative analysis of complex proteomes

An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE “classical” approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification.     

Simple on-line sample preconcentration technique for peptides based on dynamic pH junction in capillary electrophoresis-mass spectrometry

We report an on-line sample preconcentration technique based on dynamic pH junction in capillary electrophoresis-mass spectrometry (CE-MS). For peptide analysis, the samples were dissolved in a solution with higher pH than the background solution (BGS), and were injected into the capillary as a long plug. The pH difference between the sample matrix and BGS caused changes in analytes' mobilities during electrophoresis, resulting in narrowing of their bands at the boundary. Around 550-1000-fold sensitivity enhancement could be achieved in terms of peak intensity without degrading peak shape and resolution. This technique is easy to perform and will be useful for peptide mass fingerprinting in protein analysis.     

Enhancement of mass spectrometry performance for proteomic analyses using highfield asymmetric waveform ion mobility spectrometry (FAIMS)

Remarkable advances in mass spectrometry sensitivity and resolution have been accomplished over the past two decades to enhance the depth and coverage of proteome analyses. As these technological developments expanded the detection capability of mass spectrometers, they also revealed an increasing complexity of low abundance peptides, solvent clusters and sample contaminants that can confound protein identification. Separation techniques that are complementary and can be used in combination with liquid chromatography are often sought to improve mass spectrometry sensitivity for proteomics applications. In this context, high‐field asymmetric waveform ion mobility spectrometry (FAIMS), a form of ion mobility that exploits ion separation at low and high electric fields, has shown significant advantages by focusing and separating multiply charged peptide ions from singly charged interferences. This paper examines the analytical benefits of FAIMS in proteomics to separate co‐eluting peptide isomers and to enhance peptide detection and quantitative measurements of protein digests via native peptides (label‐free) or isotopically labeled peptides from metabolic labeling or chemical tagging experiments. Copyright © 2015 John Wiley & Sons, Ltd.     

Fully automated software solution for protein quantitation by global metabolic labeling with stable isotopes

Metabolic stable isotope labeling is increasingly employed for accurate protein (and metabolite) quantitation using mass spectrometry (MS). It provides sample-specific isotopologues that can be used to facilitate comparative analysis of two or more samples. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has been used for almost a decade in proteomic research and analytical software solutions have been established that provide an easy and integrated workflow for elucidating sample abundance ratios for most MS data formats. While SILAC is a discrete labeling method using specific amino acids, global metabolic stable isotope labeling using isotopes such as (15)N labels the entire element content of the sample, i.e. for (15)N the entire peptide backbone in addition to all nitrogen-containing side chains. Although global metabolic labeling can deliver advantages with regard to isotope incorporation and costs, the requirements for data analysis are more demanding because, for instance for polypeptides, the mass difference introduced by the label depends on the amino acid composition. Consequently, there has been less progress on the automation of the data processing and mining steps for this type of protein quantitation. Here, we present a new integrated software solution for the quantitative analysis of protein expression in differential samples and show the benefits of high-resolution MS data in quantitative proteomic analyses.     

A Bayesian Markov-Chain-Based Heteroscedastic Regression Model for the Analysis of <Superscript>18</Superscript>O-Labeled Mass Spectra

To reduce the influence of the between-spectra variability on the results of peptide quantification, one can consider the ¹⁸O-labeling approach. Ideally, with such labeling technique, a mass shift of 4 Da of the isotopic distributions of peptides from the labeled sample is induced, which allows one to distinguish the two samples and to quantify the relative abundance of the peptides. It is worth noting, however, that the presence of small quantities of ¹⁶O and ¹⁷O atoms during the labeling step can cause incomplete labeling. In practice, ignoring incomplete labeling may result in the biased estimation of the relative abundance of the peptide in the compared samples. A Markov model was developed to address this issue (Zhu, Valkenborg, Burzykowski. J. Proteome Res. 9, 2669–2677, 2010). The model assumed that the peak intensities were normally distributed with heteroscedasticity using a power-of-the-mean variance funtion. Such a dependence has been observed in practice. Alternatively, we formulate the model within the Bayesian framework. This opens the possibility to further extend the model by the inclusion of random effects that can be used to capture the biological/technical variability of the peptide abundance. The operational characteristics of the model were investigated by applications to real-life mass-spectrometry data sets and a simulation study.     

Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics

Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential ¹²C-/¹³C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N-Methyl-D-aspartic and tyrosine were identified. The changes of these biomarkers were likely due to the disruption of the endocrine system of silkworm by DDT. This work illustrates that the method of CIL LC-MS is useful to generate quantitative submetabolome profiles from a small volume of silkworm hemolymph with much higher coverage than conventional LC-MS methods, thereby facilitating the discovery of potential metabolite biomarkers related to EDC or other chemical exposure.     

基于保留时间和质荷比匹配的液相色谱-质谱联用技术用于非标记肽段的差异分析

建立了一种无需化学标记的,基于纳升级毛细管液相色谱-电喷雾离子阱质谱联用技术和质谱数据处理的肽段差异分析方法。本方法采用定量差异分析与肽序列鉴定分析分别进行的策略,首先对样品进行质谱全扫描的液质全谱式分析,在全扫描质谱数据中提取肽特征点信息,通过保留时间和质荷比参数匹配不同样品中的共有肽特征点,比较其相对峰强度有无差异。最后对样品中存在丰度差异的肽特征点进行选择性二级质谱分析和序列鉴定,从而实现复杂样品中肽段的差异比较分析。以血浆蛋白酶解混合物为实验对象,考察了本方法用于肽段相对定量分析的重现性以及浓度信号响应曲线等。结果表明:提取的肽特征点峰强度相对标准偏差的中值<22%,肽段离子强度动态范围达3个数量级,在5～1000fmol范围内对肽段定量具有良好线性关系。本方法可用于不同条件样品中具有倍数差异的内源性肽的比较分析。     

Comparison of LFQ and IPTL for Protein Identification and Relative Quantification

(1) Background: Mass spectrometry-based quantitative proteome profiling is most commonly performed by label-free quantification (LFQ), stable isotopic labeling with amino acids in cell culture (SILAC), and reporter ion-based isobaric labeling methods (TMT and iTRAQ). Isobaric peptide termini labeling (IPTL) was described as an alternative to these methods and is based on crosswise labeling of both peptide termini and MS2 quantification. High quantification accuracy was assumed for IPTL because multiple quantification points are obtained per identified MS2 spectrum. A direct comparison of IPTL with other quantification methods has not been performed yet because IPTL commonly requires digestion with endoproteinase Lys-C. (2) Methods: To enable tryptic digestion of IPTL samples, a novel labeling for IPTL was developed that combines metabolic labeling (Arg-0/Lys-0 and Arg-d4/Lys-d4, respectively) with crosswise N-terminal dimethylation (d4 and d0, respectively). (3) Results: The comparison of IPTL with LFQ revealed significantly more protein identifications for LFQ above homology ion scores but not above identity ion scores. (4) Conclusions: The quantification accuracy was superior for LFQ despite the many quantification points obtained with IPTL.     

Microscale solution IEF combined with 2-D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts

Current gel-based protein profiling methods such as 2-DE and fluorescent 2-D difference in gel electrophoresis (DIGE) evaluate small portions of complex proteomes. Hence, sample prefractionation is essential for more comprehensive proteome coverage and detection of low-abundant proteins. In this study, we describe the combination of DIGE labeling with microscale solution IEF (MicroSol-IEF) fractionation and subsequent analysis on slightly overlapping narrow pH range 2-D gels. By fluorescently tagging and mixing samples and controls prior to prefractionation, complications resulting from minor run-to-run variations during MicroSol-IEF separations of multiple samples are avoided. This greatly improves the reliability of quantitative comparisons. To illustrate its utility, this 3-D DIGE strategy was applied to analysis of human melanoma cells and mouse lung tissue extracts. Approximately 1000 reproducible spots can be obtained from narrow range 2-D gels of individual MicroSol-IEF fractions, and approximately 6000 spots can be obtained from entire proteomes. Quantitative changes in closely related samples could be more reliably detected and the method has a greatly increased capacity to distinguish between closely related protein isoforms. Thus the 3-D DIGE strategy produces a powerful method for more comprehensive and more reliable quantitative comparisons of protein profiles of very complex proteomes.     

Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics

Stable-isotope labeling coupled with liquid-phase separation and MS analysis is a powerful technique for comparative proteomics. We developed a dimethyl labeling strategy (Anal. Chem. 2003, 75, 6843-6852 and J. Proteome Res. 2005, 4, 101-108) to label peptide N-terminus and epsilon-amino groups of Lys with water-soluble formaldehyde via reductive methylation, and an isotopic pair of formaldehyde is used for binary labeling on two sets of samples. In this study, this approach is extended to a four sample labeling by combining the binary isotopic reagents of formaldehyde (d0, d2) and the binary isotopic reducing reagents, sodium cyanoborohydride (d0, d3). To ensure sufficient mass difference, this multiplexed labeling is coupled with endoproteinase Lys-C instead of trypsin for digestion, resulting in at least two labeling sites with a mass difference of 4 Da for each pair of peptide digest. Moreover, multiplex dimethyl labeling was proved to have no significant isotopic effect during RP LC elution. This method was further applied for monitoring Lys-C digestion using hemoglobin as a model. Dimethyl labeled digests derived from seven time points (1-30 h) were grouped into two sets of sample mixtures, separated by nano-LC to reduce the complexity, and then analyzed by ESI-MS/MS. The temporal study reveals that Lys-C digestion was completed in 10-15 h for all detected peptides. The multiplex dimethyl method has not only provided a simultaneous detection mean for four sample sets but has also conserved all the advantages associated with the original binary method.     

Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry

Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.