Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: application to glyceraldehyde-3-phosphate dehydrogenase

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.     

Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software

Tandem mass spectrometry (MS/MS) allows for the rapid identification of many types of post-translational modifications (PTMs), especially those that can be detected by a diagnostic mass shift in one or more peptide fragment ions (for example, phosphorylation). But some PTMs (for example, SUMOs and other ubiquitin-like modifiers) themselves produce multiple fragment ions; combined with fragments from the modified target peptide, a complex overlapping fragmentation pattern is thus generated, which is uninterpretable by standard peptide sequencing software. Here we introduce SUMmOn, an automated pattern recognition tool that detects diagnostic PTM fragment ion series within complex MS/MS spectra, to identify modified peptides and modification sites within these peptides. Using SUMmOn, we demonstrate for the first time that human SUMO-1 multimerizes in vitro primarily via three N-terminal lysines, Lys7, Lys16 and Lys17. Notably, our method is theoretically applicable to any type of modification or chemical moiety generating a unique fragment ion pattern.     

Optimization of apolipoprotein-B-100 sequence coverage by liquid chromatography-tandem mass spectrometry for the future study of its posttranslational modifications

Proteomic applications have been increasingly used to study posttranslational modifications of proteins (PTMs). For the purpose of identifying and localizing specific but unknown PTMs on huge proteins, improving their sequence coverage is fundamental. Using liquid chromatography coupled to mass spectrometry (LC–MS/MS), peptide mapping of the native apolipoprotein-B-100 was performed to further document the effects of oxidation. Apolipoprotein-B-100 is the main protein of low-density lipoprotein particles and its oxidation could play a role in atherogenesis. Because it is one of the largest human proteins, the sequence recovery rate of apolipoprotein-B-100 only reached 1% when conventional analysis parameters were used. The different steps of the peptide mapping process—from protein treatment to data analysis—were therefore reappraised and optimized. These optimizations allowed a protein sequence recovery rate of 79%, a rate which has never been achieved previously for such a large human protein. The key points for improving peptide mapping were optimization of the data analysis software; peptide separation by LC; sample preparation; and MS acquisition. The new protocol has allowed us to increase by a factor of 4 the detection of modified peptides in apolipoprotein-B-100. This approach could easily be transferred to any study of PTMs using LC–MS/MS.     

MMS2plot: An R Package for Visualizing Multiple MS/MS Spectra for Groups of Modified and Non‐Modified Peptides

A large number of post‐translational modifications (PTMs) in proteins are buried in the unassigned mass spectrometric (MS) spectra in shot‐gun proteomics datasets. Because the modified peptide fragments are low in abundance relative to the corresponding non‐modified versions, it is critical to develop tools that allow facile evaluation of assignment of PTMs based on the MS/MS spectra. Such tools will preferably have the ability to allow comparison of fragment ion spectra and retention time between the modified and unmodified peptide pairs or group. Herein, MMS2plot, an R package for visualizing peptide‐spectrum matches (PSMs) for multiple peptides, is described. MMS2plot features a batch mode and generates the output images in vector graphics file format that facilitate evaluation and publication of the PSM assignment. MMS2plot is expected to play an important role in PTM discovery from large‐scale proteomics datasets generated by liquid chromatography‐MS/MS. The MMS2plot package is freely available at https://github.com/lileir/MMS2plot under the GPL‐3 license.     

P-Mod: an algorithm and software to map modifications to peptide sequences using tandem MS data

The discovery of unanticipated protein modifications is one of the most challenging problems in proteomics. Whereas widely used algorithms such as Sequest and Mascot enable mapping of modifications when the mass and amino acid specificity are known, unexpected modifications cannot be identified with these tools. We have developed an algorithm and software called P-Mod, which enables discovery and sequence mapping of modifications to target proteins known to be represented in the analysis or identified by Sequest. P-Mod matches MS/MS spectra to peptide sequences in a search list. For spectra of modified peptides, P-Mod calculates mass differences between search peptide sequences and MS/MS precursors and localizes the mass shift to a sequence position in the peptide. Because modifications are detected as mass shifts, P-Mod does not require the user to guess at masses or sequence locations of modifications. P-Mod uses extreme value statistics to assign p value estimates to sequence-to-spectrum matches. The reported p values are scaled to account for the number of comparisons, so that error rates do not increase with the expanded search lists that result from incorporating potential peptide modifications. Combination of P-Mod searches from multiple LC-MS/MS analyses and multiple samples revealed previously unreported BSA modifications, including a novel decarboxymethylation or D-->G substitution at position 579 of the protein. P-Mod can serve a unique role in the identification of protein modifications both from exogenous and endogenous sources and may be useful for identifying modified protein forms as biomarkers for toxicity and disease processes.     

IPEP: an in silico tool to examine proteolytic peptides for mass spectrometry

Peptide-based proteomics supports identification and quantification as well as localization of post-translational modifications (PTMs) within proteins extracted from biological samples. The 'bottom-up' approach involves the digestion of proteins into peptide fragments that can be detected and sequenced with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A web-based application, iPEP, was developed to compare the effectiveness of different proteolytic digests in detecting specific sequences. Furthermore, peptide populations can be examined to help optimize detection of certain groups of proteins relative to the proteome and the digested peptidome. The application reports proteolytic peptide sequences, theoretical molecular weights and functional annotations using Gene Ontology (GO) terms. The iPEP tool can assist with experimental design by maximizing the detection of proteins, consensus sites and modified residues of interest for individual proteins or as part of large-scale proteomic assays. AVAILABILITY: http://ipep.moffitt.org     

VEMS 3.0: algorithms and computational tools for tandem mass spectrometry based identification of post-translational modifications in proteins

Protein and peptide mass analysis and amino acid sequencing by mass spectrometry is widely used for identification and annotation of post-translational modifications (PTMs) in proteins. Modification-specific mass increments, neutral losses or diagnostic fragment ions in peptide mass spectra provide direct evidence for the presence of post-translational modifications, such as phosphorylation, acetylation, methylation or glycosylation. However, the commonly used database search engines are not always practical for exhaustive searches for multiple modifications and concomitant missed proteolytic cleavage sites in large-scale proteomic datasets, since the search space is dramatically expanded. We present a formal definition of the problem of searching databases with tandem mass spectra of peptides that are partially (sub-stoichiometrically) modified. In addition, an improved search algorithm and peptide scoring scheme that includes modification specific ion information from MS/MS spectra was implemented and tested using the Virtual Expert Mass Spectrometrist (VEMS) software. A set of 2825 peptide MS/MS spectra were searched with 16 variable modifications and 6 missed cleavages. The scoring scheme returned a large set of post-translationally modified peptides including precise information on modification type and position. The scoring scheme was able to extract and distinguish the near-isobaric modifications of trimethylation and acetylation of lysine residues based on the presence and absence of diagnostic neutral losses and immonium ions. In addition, the VEMS software contains a range of new features for analysis of mass spectrometry data obtained in large-scale proteomic experiments. Windows binaries are available at http://www.yass.sdu.dk/.     

Identification of post-translational modifications by blind search of mass spectra

Most tandem mass spectrometry (MS/MS) database search algorithms perform a restrictive search that takes into account only a few types of post-translational modifications (PTMs) and ignores all others. We describe an unrestrictive PTM search algorithm, MS-Alignment, that searches for all types of PTMs at once in a blind mode, that is, without knowing which PTMs exist in nature. Blind PTM identification makes it possible to study the extent and frequency of different types of PTMs, still an open problem in proteomics. Application of this approach to lens proteins resulted in the largest set of PTMs reported in human crystallins so far. Our analysis of various MS/MS data sets implies that the biological phenomenon of modification is much more widespread than previously thought. We also argue that MS-Alignment reveals some uncharacterized modifications that warrant further experimental validation.     

The utility of ETD mass spectrometry in proteomic analysis

Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.     

Fast multi-blind modification search through tandem mass spectrometry

With great biological interest in post-translational modifications (PTMs), various approaches have been introduced to identify PTMs using MS/MS. Recent developments for PTM identification have focused on an unrestrictive approach that searches MS/MS spectra for all known and possibly even unknown types of PTMs at once. However, the resulting expanded search space requires much longer search time and also increases the number of false positives (incorrect identifications) and false negatives (missed true identifications), thus creating a bottleneck in high throughput analysis. Here we introduce MODa, a novel "multi-blind" spectral alignment algorithm that allows for fast unrestrictive PTM searches with no limitation on the number of modifications per peptide while featuring over an order of magnitude speedup in relation to existing approaches. We demonstrate the sensitivity of MODa on human shotgun proteomics data where it reveals multiple mutations, a wide range of modifications (including glycosylation), and evidence for several putative novel modifications. Based on the reported findings, we argue that the efficiency and sensitivity of MODa make it the first unrestrictive search tool with the potential to fully replace conventional restrictive identification of proteomics mass spectrometry data.     

Enhanced detection and identification of multiply phosphorylated peptides using TiO2 enrichment in combination with MALDI TOF/TOF MS

The analysis of PTMs such as phosphorylation has become an important field in MS because they can directly indicate protein states and interactions. Whereas the characterization of singly and doubly phosphorylated peptides has almost become routine, identifying phosphorylation events at multiple residues within a small region of a protein is still problematic. The identification of multiple modifications can be further hampered by low sequence information due to multiple neutral losses from phosphorylated side chains. Here we present a strategy for the analysis of complex phosphopeptides that combines peptide enrichment by titanium dioxide, separation by RP separation on monolithic columns and MS using high energy HE-CAD in a MALDI TOF/TOF analyser. Using synthetic phosphopeptides our approach is compared to multistage activation (MSA) MS/MS and the recently described electron transfer dissociation (ETD) method using an ESI-LTQ mass spectrometer.     

Comprehensive identification of novel post-translational modifications in cellular peroxiredoxin 6

Peroxiredoxin 6 (PRDX6), a 1-Cys peroxiredoxin, is a bifunctional enzyme acting both as a glutathione peroxidase and a phospholipase A2. However, the underlying mechanisms and their regulation mechanisms are not well understood. Because post-translational modifications (PTMs) have been shown to play important roles in the function of many proteins, we undertook, in this study, to identify the PTMs in PRDX6 utilizing proteomic tools including nanoUPLC-ESI-q-TOF MS/MS employing selectively excluded mass screening analysis (SEMSA) in conjunction with MOD(i) and MODmap algorithm. We chose PRDX6 obtained from liver tissues from two inbred mouse strains, C57BL/6J and C3H/HeJ, which vary in their susceptibility to high-fat diet-induced obesity and atherosclerosis, and a B16F10 melanoma cell line for this study. When PRDX6 protein samples were separated on 2D-PAGE based on pI, several PRDX6 spots appeared. They were purified and the low abundant PTMs in each PRDX6 spot were analyzed. Unexpected mass shifts (Δm = -34, +25, +64, +87, +103, +134, +150, +284 Da) observed at active site cysteine residue (Cys47) were quantified using precursor ion intensities. Mass differences of -34, +25, and +64 Da are presumed to reflect the conversion of cysteine to dehydroalanine, cyano, and Cys-SO(2) -SH, respectively. We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da) as well as unknown modifications (+134, +150, +284 Da). Comprehensive analysis of these PTMs revealed that the PRDX6 exists as a heterogeneous mixture of molecules containing a multitude of PTMs. Several of these modifications occur at cysteine residue in the enzyme active site. Other modifications observed, in PRDX6 from mouse liver tissues included, among others, mono- and dioxidation at Trp and Met, acetylation at Lys, and deamidation at Asn and Gln. Comprehensive identification of the diverse PTMs occurring in this bifunctional PRDX6 enzyme should help understand how PRDX6 plays key roles in oxidative stresses.     

Analysis of posttranslational modifications of proteins by tandem mass spectrometry

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.     

Monoclonal antibodies against pools of mono- and polyacetylated peptides selectively recognize acetylated lysines within the context of the original antigen

Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24–39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.     

A new method for C-terminal sequence analysis in the proteomic era

The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the amino or carboxy terminus are by far the most common types of PTMs in proteins. In contrast to the analysis of phosphorylation and glycosylation sites, relatively little attention has been paid to the development of approaches for the systematic analysis of proteolytic processing events. Here we present a new mass spectrometry (MS)-based strategy that allows the identification of the C-terminal sequence of proteins. The method can be directly applied to proteins cleaved with cyanogen bromide (CNBr) and purified either by SDS-PAGE, by two-dimensional (2D) PAGE or in solution, and it therefore eliminates the need for specific isolation of the C-terminal peptide. Using Shewanella oneidensis as a model system, we have demonstrated that this approach can be used for C-terminal sequence analysis at a proteomic scale. We also applied the method to study the C-terminal proteolytic processing of procardosin A.     

Gaining knowledge from previously unexplained spectra-application of the PTM-Explorer software to detect PTM in HUPO BPP MS/MS data

A novel software tool named PTM-Explorer has been applied to LC-MS/MS datasets acquired within the Human Proteome Organisation (HUPO) Brain Proteome Project (BPP). PTM-Explorer enables automatic identification of peptide MS/MS spectra that were not explained in typical sequence database searches. The main focus was detection of PTMs, but PTM-Explorer detects also unspecific peptide cleavage, mass measurement errors, experimental modifications, amino acid substitutions, transpeptidation products and unknown mass shifts. To avoid a combinatorial problem the search is restricted to a set of selected protein sequences, which stem from previous protein identifications using a common sequence database search. Prior to application to the HUPO BPP data, PTM-Explorer was evaluated on excellently manually characterized and evaluated LC-MS/MS data sets from Alpha-A-Crystallin gel spots obtained from mouse eye lens. Besides various PTMs including phosphorylation, a wealth of experimental modifications and unspecific cleavage products were successfully detected, completing the primary structure information of the measured proteins. Our results indicate that a large amount of MS/MS spectra that currently remain unidentified in standard database searches contain valuable information that can only be elucidated using suitable software tools.     

Protein mass analysis of histones

Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.     

The Plant PTM Viewer,a central resource for exploring plant protein modifications

Post‐translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site‐specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370 000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows the submission of mass spectrometry‐based PTM data to remain at pace with future PTM plant studies.     

Biologically and diagenetically derived peptide modifications in moa collagens

The modifications that occur on proteins in natural environments over time are not well studied, yet characterizing them is vital to correctly interpret sequence data recovered from fossils. The recently extinct moa (Dinornithidae) is an excellent candidate for investigating the preservation of proteins, their post-translational modifications (PTMs) and diagenetic alterations during degradation. Moa protein extracts were analysed using mass spectrometry, and peptides from collagen I, collagen II and collagen V were identified. We also identified biologically derived PTMs (i.e. methylation, di-methylation, alkylation, hydroxylation, fucosylation) on amino acids at locations consistent with extant proteins. In addition to these in vivo modifications, we detected novel modifications that are probably diagenetically derived. These include loss of hydroxylation/glutamic semialdehyde, carboxymethyllysine and peptide backbone cleavage, as well as previously noted deamidation. Moa collagen sequences and modifications provide a baseline by which to evaluate proteomic studies of other fossils, and a framework for defining the molecular relationship of moa to other closely related taxa.     

Data-independent acquisition proteomics methods for analyzing post-translational modifications

Protein post-translational modifications (PTMs) increase the functional diversity of the cellular proteome. Accurate and high throughput identification and quantification of protein PTMs is a key task in proteomics research. Recent advancements in data-independent acquisition (DIA) mass spectrometry (MS) technology have achieved deep coverage and accurate quantification of proteins and PTMs. This review provides an overview of DIA data processing methods that cover three aspects of PTMs analysis, that is, detection of PTMs, site localization, and characterization of complex modification moieties, such as glycosylation. In addition, a survey of deep learning methods that boost DIA-based PTMs analysis is presented, including in silico spectral library generation, as well as feature scoring and error rate control. The limitations and future directions of DIA methods for PTMs analysis are also discussed. Novel data analysis methods will take advantage of advanced MS instrumentation techniques to empower DIA MS for in-depth and accurate PTMs measurements.