PTMD: A Database of Human Disease-associated Post-translational Modifications

Various posttranslational modifications(PTMs) participate in nearly all aspects of biological processes by regulating protein functions, and aberrant states of PTMs are frequently implicated in human diseases. Therefore, an integral resource of PTM–disease associations(PDAs)would be a great help for both academic research and clinical use. In this work, we reported PTMD,a well-curated database containing PTMs that are associated with human diseases. We manually collected 1950 known PDAs in 749 proteins for 23 types of PTMs and 275 types of diseases from the literature. Database analyses show that phosphorylation has the largest number of disease associations, whereas neurologic diseases have the largest number of PTM associations. We classified all known PDAs into six classes according to the PTM status in diseases and demonstrated that the upregulation and presence of PTM events account for a predominant proportion of diseaseassociated PTM events. By reconstructing a disease–gene network, we observed that breast cancershave the largest number of associated PTMs and AKT1 has the largest number of PTMs connected to diseases. Finally, the PTMD database was developed with detailed annotations and can be a useful resource for further analyzing the relations between PTMs and human diseases. PTMD is freely accessible at http://ptmd.biocuckoo.org.     

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.     

Functional analysis tools for post‐translational modification: a post‐translational modification database for analysis of proteins and metabolic pathways

Post‐translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high‐resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post‐Translational Modifications (FAT‐PTM) database ( https://bioinformatics.cse.unr.edu/fat-ptm/ ), which currently supports eight different types of PTM and over 49 000 PTM sites identified in large‐scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT‐PTM database currently supports tools to visualize protein‐centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein‐centric metabolic pathways and groups of proteins that are co‐modified by multiple PTMs. Overall, the FAT‐PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.     

Rampant purifying selection conserves positions with posttranslational modifications in human proteins

Posttranslational modifications (PTMs) are chemical alterations that are critical to protein conformation and activation states. Despite their functional importance and reported involvement in many diseases, evolutionary analyses have produced enigmatic results because only weak or no selective pressures have been attributed to many types of PTMs. In a large-scale analysis of 16,836 PTM positions from 4,484 human proteins, we find that positions harboring PTMs show evidence of higher purifying selection in 70% of the phosphorylated and N-linked glycosylated proteins. The purifying selection is up to 42% more severe at PTM residues as compared with the corresponding unmodified amino acids. These results establish extensive selective pressures in the long-term history of positions that experience PTMs in the human proteins. Our findings will enhance our understanding of the historical function of PTMs over time and help in predicting PTM positions by using evolutionary comparisons.     

Proteomics of post-translational modifications in colorectal cancer: Discovery of new biomarkers

Colorectal cancer (CRC) is one of the costliest health problems and ranks second in cancer-related mortality in developed countries. With the aid of proteomics, many protein biomarkers for the diagnosis, prognosis, and precise management of CRC have been identified. Furthermore, some protein biomarkers exhibit structural diversity after modifications. Post-translational modifications (PTMs), most of which are catalyzed by a variety of enzymes, extensively increase protein diversity and are involved in many complex and dynamic cellular processes through the regulation of protein function. Accumulating evidence suggests that abnormal PTM events are associated with a variety of human diseases, such as CRC, thus highlighting the need for studying PTMs to discover both the molecular mechanisms and therapeutic targets of CRC. In this review, we begin with a brief overview of the importance of protein PTMs, discuss the general strategies for proteomic profiling of several key PTMs (including phosphorylation, acetylation, glycosylation, ubiquitination, methylation, and citrullination), shift the emphasis to describing the specific methods used for delineating the global landscapes of each of these PTMs, and summarize the recent applications of these methods to explore the potential roles of the PTMs in CRC. Finally, we discuss the current status of PTM research on CRC and provide future perspectives on how PTM regulation can play an essential role in translational medicine for early diagnosis, prognosis stratification, and therapeutic intervention in CRC.     

An atlas of posttranslational modifications on RNA binding proteins

RNA structure and function are intimately tied to RNA binding protein recognition and regulation. Posttranslational modifications are chemical modifications which can control protein biology. The role of PTMs in the regulation RBPs is not well understood, in part due to a lacking analysis of PTM deposition on RBPs. Herein, we present an analysis of posttranslational modifications (PTMs) on RNA binding proteins (RBPs; a PTM RBP Atlas). We curate published datasets and primary literature to understand the landscape of PTMs and use protein–protein interaction data to understand and potentially provide a framework for understanding which enzymes are controlling PTM deposition and removal on the RBP landscape. Intersection of our data with The Cancer Genome Atlas also provides researchers understanding of mutations that would alter PTM deposition. Additional characterization of the RNA–protein interface provided from in-cell UV crosslinking experiments provides a framework for hypotheses about which PTMs could be regulating RNA binding and thus RBP function. Finally, we provide an online database for our data that is easy to use for the community. It is our hope our efforts will provide researchers will an invaluable tool to test the function of PTMs controlling RBP function and thus RNA biology.     

Diurnal changes in concerted plant protein phosphorylation and acetylation in Arabidopsis organs and seedlings

Protein phosphorylation and acetylation are the two most abundant post‐translational modifications (PTMs) that regulate protein functions in eukaryotes. In plants, these PTMs have been investigated individually; however, their co‐occurrence and dynamics on proteins is currently unknown. Using Arabidopsis thaliana, we quantified changes in protein phosphorylation, acetylation and protein abundance in leaf rosettes, roots, flowers, siliques and seedlings at the end of day (ED) and at the end of night (EN). This identified 2549 phosphorylated and 909 acetylated proteins, of which 1724 phosphorylated and 536 acetylated proteins were also quantified for changes in PTM abundance between ED and EN. Using a sequential dual‐PTM workflow, we identified significant PTM changes and intersections in these organs and plant developmental stages. In particular, cellular process‐, pathway‐ and protein‐level analyses reveal that the phosphoproteome and acetylome predominantly intersect at the pathway‐ and cellular process‐level at ED versus EN. We found 134 proteins involved in core plant cell processes, such as light harvesting and photosynthesis, translation, metabolism and cellular transport, that were both phosphorylated and acetylated. Our results establish connections between PTM motifs, PTM catalyzing enzymes and putative substrate networks. We also identified PTM motifs for further characterization of the regulatory mechanisms that control cellular processes during the diurnal cycle in different Arabidopsis organs and seedlings. The sequential dual‐PTM analysis expands our understanding of diurnal plant cell regulation by PTMs and provides a useful resource for future analyses, while emphasizing the importance of analyzing multiple PTMs simultaneously to elucidate when, where and how they are involved in plant cell regulation.     

Analysis of the role of protein phosphorylation in the development of diseases

During recent decades significant progress in studies of the molecular basis of socially significant diseases has been achieved due to introduction of high-throughput methods of genomics and proteomics. Numerous studies, performed within the global program “Human Proteome,” were aimed at identifying all possible proteins in various (including cancer) cell cultures and tissues. One of the aims was to identify socalled biomarkers—the proteins, specific for certain pathologies. However, many studies have shown that the development of the disease is not associated with appearance of new proteins, but it depends on the expression level of certain genes or specific proteoforms representing splice variants, single amino acid polymorphism (SAP) and post-translational modifications (PTM) of proteins. PTMs can play a key role in the development of pathology, because they activate various regulatory or structural proteins in most cellular processes. Among such modifications, phosphorylation appears to be the most significant PTM. This review considers methods of analysis of protein phosphorylation used in studies of the molecular basis of oncological diseases; it contains examples illustrating contribution of modified proteins directly involved in their development as well as examples of screening of such crucial PTMs in diagnostics and selection of methods for treatment.     

The structural context of posttranslational modifications at a proteome-wide scale

The recent revolution in computational protein structure prediction provides folding models for entire proteomes, which can now be integrated with large-scale experimental data. Mass spectrometry (MS)-based proteomics has identified and quantified tens of thousands of posttranslational modifications (PTMs), most of them of uncertain functional relevance. In this study, we determine the structural context of these PTMs and investigate how this information can be leveraged to pinpoint potential regulatory sites. Our analysis uncovers global patterns of PTM occurrence across folded and intrinsically disordered regions. We found that this information can help to distinguish regulatory PTMs from those marking improperly folded proteins. Interestingly, the human proteome contains thousands of proteins that have large folded domains linked by short, disordered regions that are strongly enriched in regulatory phosphosites. These include well-known kinase activation loops that induce protein conformational changes upon phosphorylation. This regulatory mechanism appears to be widespread in kinases but also occurs in other protein families such as solute carriers. It is not limited to phosphorylation but includes ubiquitination and acetylation sites as well. Furthermore, we performed three-dimensional proximity analysis, which revealed examples of spatial coregulation of different PTM types and potential PTM crosstalk. To enable the community to build upon these first analyses, we provide tools for 3D visualization of proteomics data and PTMs as well as python libraries for data accession and processing.

A combination of the comprehensive structural predictions of AlphaFold2 and large-scale proteomics data on post-translational modifications (PTMs) reveals novel insights into the functional importance of PTMs, based on their structural context.     

An emerging field: Post-translational modification in microbiome

Post-translational modifications (PTMs) play an essential role in most biological processes. PTMs on human proteins have been extensively studied. Studies on bacterial PTMs are emerging, which demonstrate that bacterial PTMs are different from human PTMs in their types, mechanisms and functions. Few PTM studies have been done on the microbiome. Here, we reviewed several studied PTMs in bacteria including phosphorylation, acetylation, succinylation, glycosylation, and proteases. We discussed the enzymes responsible for each PTM and their functions. We also summarized the current methods used to study microbiome PTMs and the observations demonstrating the roles of PTM in the microbe-microbe interactions within the microbiome and their interactions with the environment or host. Although new methods and tools for PTM studies are still needed, the existing technologies have made great progress enabling a deeper understanding of the functional regulation of the microbiome. Large-scale application of these microbiome-wide PTM studies will provide a better understanding of the microbiome and its roles in the development of human diseases.     

Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays

Background

Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens.

Methodology/Principal Findings

In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay.

Conclusions/Significance

This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research.     

Post-translational modification of plant-made foreign proteins; glycosylation and beyond

The complex and diverse nature of the post-translational modification (PTM) of proteins represents an efficient and cost-effective mechanism for the exponential diversification of the genome. PTMs have been shown to affect almost every aspect of protein activity, including function, localisation, stability, and dynamic interactions with other molecules. Although many PTMs are evolutionarily conserved there are also important kingdom-specific modifications which should be considered when expressing recombinant proteins. Plants are gaining increasing acceptance as an expression system for recombinant proteins, particularly where eukaryotic-like PTMs are required. Glycosylation is the most extensively studied PTM of plant-made recombinant proteins. However, other types of protein processing and modification also occur which are important for the production of high quality recombinant protein, such as hydroxylation and lipidation. Plant and/or protein engineering approaches offer many opportunities to exploit PTM pathways allowing the molecular farmer to produce a humanised product with modifications functionally similar or identical to the native protein. Indeed, plants have demonstrated a high degree of tolerance to changes in PTM pathways allowing recombinant proteins to be modified in a specific and controlled manner, frequently resulting in a homogeneity of product which is currently unrivalled by alternative expression platforms. Whether a recombinant protein is intended for use as a scientific reagent, a cosmetic additive or as a pharmaceutical, PTMs through their presence and complexity, offer an extensive range of options for the rational design of humanised (biosimilar), enhanced (biobetter) or novel products.     

Deciphering a global network of functionally associated post‐translational modifications

Various post‐translational modifications (PTMs) fine‐tune the functions of almost all eukaryotic proteins, and co‐regulation of different types of PTMs has been shown within and between a number of proteins. Aiming at a more global view of the interplay between PTM types, we collected modifications for 13 frequent PTM types in 8 eukaryotes, compared their speed of evolution and developed a method for measuring PTM co‐evolution within proteins based on the co‐occurrence of sites across eukaryotes. As many sites are still to be discovered, this is a considerable underestimate, yet, assuming that most co‐evolving PTMs are functionally associated, we found that PTM types are vastly interconnected, forming a global network that comprise in human alone >50 000 residues in about 6000 proteins. We predict substantial PTM type interplay in secreted and membrane‐associated proteins and in the context of particular protein domains and short‐linear motifs. The global network of co‐evolving PTM types implies a complex and intertwined post‐translational regulation landscape that is likely to regulate multiple functional states of many if not all eukaryotic proteins.     

Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.Post-translational modifications (PTMs)¹ are a rapidly expanding and important class of protein feature that broaden the functional diversity of proteins in a proteome. By definition, PTMs change protein structure and therefore have the potential to affect protein function by altering protein interactions, protein stability or catalytic activity (1, 2). As they have been found to occur on nearly every protein in the eukaryotic proteome, PTMs broadly impact nearly all known cellular processes. Over 300 different types of PTM are known, ranging from single atom modifications (e.g. oxide) to small protein modifiers (e.g. ubiquitin), which can occur on all but five amino acid residues resulting from enzymatic or nonenzymatic processes (3). Over 220,000 distinct PTM sites have been experimentally identified across ∼77,000 different proteins to date (dbPTM; http://dbptm.mbc.nctu.edu.tw/statistics.php) – numbers that continue to grow exponentially because of improved methods for high throughput detection by mass spectrometry (MS). By virtue of how they are detected, most PTM data are sequence-linked and lack structural context.The function of most PTMs is unknown because the rate of PTM detection far surpasses the rate at which any one modification can be studied empirically. Moreover, the functional impact of every PTM is likely not equivalent (4). For example, computational analysis of phosphorylation sites in yeast and human proteomes indicate that well-conserved phosphosites are more likely to have a functional consequence compared with poorly conserved sites, yet only a fraction of phosphosites are well conserved (5, 6). Consequently, the development of tools that provide functional prioritization of PTMs could have a broad impact on our understanding of protein regulation, biological mechanism, and molecular evolution.The emerging need for methods that predict the functional impact of a PTM has not yet been met. Longstanding methods capitalize predominantly on the sequence context of PTMs and have been used to predict sites of modification (expasy.org/proteomics/post-translational_modification) and to compare enzyme/substrate interactions (7–9). More recently, studies aimed at expanding the parameters associated with functional PTMs have emerged. In these cases, a set of common features correlated with functional importance are derived from the analysis of PTMs within and between organisms including: number of PTM observations at a multiple sequence alignment position (i.e. hotspots), measures of co-occurrence between different PTMs (e.g. distance between phosphorylation and ubiquitination sites), biological dynamics (up or down-regulation), and protein–protein interaction influence (7, 10–12). Recent efforts to provide structural context by linking individual PTMs to three-dimensional structures in the protein data bank (PDB) have also been described (13, 14). However, these resources are extensions of existing PTM databases that allow visualization of single instances of modification onto individual proteins, but do not provide quantitative or analytical value.In principle, combining PTM hotspot and structural analysis would offer multiple advantages over any one approach used in isolation. Sequence homology provides protein family membership—thereby clustering PTMs into hotspots for groups of proteins to provide information about: (1) the evolutionary conservation and (2) observation frequencies of PTMs within the family. A primary consequence of their sequence homology is that members of a protein family will exhibit similar structures and protein interactions—features that dictate the function of protein systems. A secondary consequence is that PTM hotspots generated by alignment can be projected onto family-representative protein structures, which places each PTM hotspot into a three-dimensional context that can be visualized for each family. The structural context enabled by this projection can also provide spatial information about the PTM site that can supplement the sequence characteristics of the hotspot, namely: (3) solvent accessibility, which provides an estimate of whether a modification could occur on the folded protein; and (4) protein interface residence, which indicates the potential of the PTM to disrupt protein–protein interactions. Despite the theoretical advantages, no single tool has been developed that exploits the quantitative output from both sequence and structural data to evaluate the function potential of PTMs.Here we describe a new analytical method – Structural Analysis of PTM Hotspots (SAPH-ire), which ranks PTM hotspots by their potential to impact biological function for distinct protein families (Fig. 1). We demonstrate the application of SAPH-ire to the complete set of PTMs for eight distinct protein families including large heterotrimeric G proteins—revealing high-ranking hotspots for which a biological function has not yet been determined. In particular, SAPH-ire revealed the N-terminal tail (Nt) of G protein gamma (Gγ) subunits as one of the highest ranking PTM hotspots for heterotrimeric G proteins (Gα, Gβ, and Gγ). We tested this prediction by monitoring the phosphorylation state and mutation effects of phosphorylation sites in the N terminus of the yeast Gγ subunit (Ste18). Consistent with SAPH-ire predictions, we found that phosphorylation of Ste18-Nt is biologically responsive to a GPCR stimulus and that phospho-null or phospho-mimic mutation of these sites controls protein abundance in an opposite manner in vivo. Thus, SAPH-ire is a powerful new method for predicting the function potential of PTM hotspots, which can guide empirical research toward the discovery of new protein regulatory elements based on high-throughput proteomics.Open in a separate windowFig. 1.Schematic diagram of the SAPH-ire method. A, SAPH-ire integrates InterPro, the Protein Data bank (PDB) and a customized database of experimentally validated PTMs. Uniprot entries with PTMs that belong to specific InterPro-classified protein families undergo multiple-sequence alignment (MSA) and PTM hotspot analysis (HSA), which layers all PTMs for a given alignment position in the MSA. The total PTMs observed in each hotspot and the conservation of a modifiable residue (e.g. conservation lysine at a ubiquitination hotspot) at the hotspot are quantified. B, PTM hotspots within the protein family are then projected onto all known crystal structures for the family using the Structural Projection of PTMs (SPoP) tool. From the structural topology of PTM hotspots generated by SPoP, the solvent accessible surface area (SASA) and protein interface residence is quantified for each hotspot. C, PTM Function Potential Calculator (FPC) integrates the output from HSA and SPoP, resulting in PTM function potential scores for each hotspot. The function potential score can be used to rank PTM hotspots within or between protein families – prioritizing hotspots with the greatest potential to be biologically regulated and/or effect a biological function for the protein family of interest.     

Post-translational quantitation by SRM/MRM: applications in cardiology

Introduction: Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization, and interaction with other molecules. PTMs apply a dynamic control of proteins in both physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution mass spectrometry (MS)/MS analysis provide attractive results on PTM characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples.

Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported, and a specific section is focused on PTM detection in proteins that are involved in the cardiovascular system and heart diseases.

Expert commentary: PTM characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded our knowledge in the last few years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTM complexity. Promising studies confirm the great potential of SRM/MRM to study PTMs in the cardiovascular field, and PTMomics could be very useful in the clinical perspective.     

Systematic assessment of the benefits and caveats in mining microbial post-translational modifications from shotgun proteomic data: the response of Shewanella oneidensis to chromate exposure

Microbes are known to regulate both gene expression and protein activity through the use of post-translational modifications (PTMs). Common PTMs involved in cellular signaling and gene control include methylations, acetylations, and phosphorylations, whereas oxidations have been implicated as an indicator of stress. Shewanella oneidensis MR-1 is a Gram-negative bacterium that demonstrates both respiratory versatility and the ability to sense and adapt to diverse environmental conditions. The data set used in this study consisted of tandem mass spectra derived from midlog phase aerobic cultures of S. oneidensis either native or shocked with 1 mM chromate [Cr(VI)]. In this study, three algorithms (DBDigger, Sequest, and InsPecT) were evaluated for their ability to scrutinize shotgun proteomic data for evidence of PTMs. The use of conservative scoring filters for peptides or proteins versus creating a subdatabase first from a nonmodification search was evaluated with DBDigger. The use of higher-scoring filters for peptide identifications was found to result in optimal identifications of PTM peptides with a 2% false discovery rate (FDR) for the total data set using the DBDigger algorithm. However, the FDR climbs to unacceptably high levels when only PTM peptides are considered. Sequest was evaluated as a method for confirming PTM peptides putatively identified using DBDigger; however, there was a low identification rate ( approximately 25%) for the searched spectra. InsPecT was found to have a much lower, and thus more acceptable, FDR than DBDigger for PTM peptides. Comparisons between InsPecT and DBDigger were made with respect to both the FDR and PTM peptide identifications. As a demonstration of this approach, a number of S. oneidensis chemotaxis proteins as well as low-abundance signal transduction proteins were identified as being post-translationally modified in response to chromate challenge.