Identification Of Post-translational Modifications Research Articles

Demystifying PTM Identification Using MODplus: Best Practices and Pitfalls.

Tandem mass spectrometry (MS/MS) facilitates the rapid identification of posttranslational modifications (PTMs), which play a pivotal role in regulating numerous biological processes. This chapter explores recent advancements that expand the types of detectable PTMs and enhance the speed of the PTM searches. We also delve into computational challenges associated with searching for a multitude of PTMs simultaneously. The latter section introduces an automated procedure to identify an extensive range of PTMs using MODplus, a free PTM analysis software tool. We guide the reader through the preparation of the modification search, the determination of optional search parameters, the execution of the search, and the analysis of results, exemplified by a case study using specific MS/MS dataset.

Influence of pneumatic transportation on the stability of monoclonal antibodies

Pneumatic transportation systems (PTS) were recently proposed as a method to carry ready-for-injection diluted monoclonal antibodies (mAbs) from the pharmacy to the bedside of patients. This method reduces transportation time and improves the efficiency of drug distribution process. However, mAbs are highly sensitive molecules for which subtle alterations may lead to deleterious clinical effects. These alterations can be caused by various external factors such as temperature, pH, pressure, and mechanical forces that may occur during transportation. Hence, it is essential to ensure that the mAbs transported by PTS remain stable and active throughout the transportation process. This study aims to determine the safety profile of PTS to transport 11 routinely used mAbs in a clinical setting through assessment of critical quality attributes (CQA) and orthogonal analysis. Hence, we performed aggregation/degradation profiling, post-translational modifications identification using complementary mass spectrometry-based methods, along with visible and subvisible particle formation determination by light absorbance and light obscuration analysis. Altogether, these results highlight that PTS can be safely used for this purpose when air is removed from the bags during preparation.

An Optimized and High-Throughput Method for Histone Propionylation and Data-Independent Acquisition Analysis for the Identification and Quantification of Histone Post-translational Modifications.

Histones are DNA binding proteins that allow for packaging of the DNA into the nucleus. They are abundantly present across the genome and thus serve as a major site of epigenetic regulation through the use of post-translational modifications (PTMs). Aberrations in histone expression and modifications have been implicated in a variety of human diseases and thus are a major focus of disease etiology studies. A well-established method for studying histones and PTMs is through the chemical derivatization of isolated histones followed by liquid chromatography and mass spectrometry analysis. Using such an approach has allowed for a swath of discoveries to be found, leading to novel therapeutics such as histone deacetylase (HDAC) inhibitors that have already been applied in the clinic. However, with the rapid improvement in instrumentation and data analysis pipelines, it remains important to temporally re-evaluate the established protocols to improve throughput and ensure data quality. Here, we optimized the histone derivatization procedure to increase sample throughput without compromising peptide quantification. An implemented spike-in standard peptide further serves as a quality control to evaluate the propionylation and digestion efficiencies as well as reproducibility in chromatographic retention and separation. Last, the application of various data-independent acquisition (DIA) strategies was explored to ensure low variation between runs. The output of this study is a newly optimized derivatization protocol and mass spectrometry method that maintains high identification and quantification of histone PTMs while increasing sample throughput.

New Strategies for Probing the Biological Functions of Protein Post-translational Modifications in Mammalian Cells with Genetic Code Expansion.

Protein post-translational modification (PTM) is a major mechanism for functional diversification of the human genome and plays a crucial role in almost every aspect of cellular processes, and the dysregulation of the protein PTM network has been associated with a variety of human diseases. Using high-resolution mass spectrometry, protein PTMs can be efficiently discovered and profiled under various biological and physiological conditions. However, it is often challenging to address the biological function of PTMs with biochemical and mutagenesis-based approaches. Specifically, this field lacks methods that allow gain-of-function studies of protein PTMs to understand their functional consequences in living cells. In this context, the genetic code expansion (GCE) strategy has made tremendous progress in the direct installation of PTMs and their analogs in the form of noncanonical amino acids (ncAAs) for gain-of-function investigations.In addition to studying the biological functions of known protein PTMs, the discovery of new protein PTMs is even more challenging due to the lack of chemical information for designing specific enrichment methods. Genetically encoded ncAAs in the proteome can be used as specific baits to enrich and subsequently identify new PTMs by mass spectrometry.In this Account, we discuss recent developments in the investigation of the biological functions of protein PTMs and the discovery of protein PTMs using new GCE strategies. First, we leveraged a chimeric design to construct several broadly orthogonal translation systems (OTSs). These broad OTSs can be engineered to efficiently incorporate different ncAAs in both E. coli and mammalian cells. With these broad OTSs, we accomplish the following: (1) We develop a computer-aided strategy for the design and genetic incorporation of length-tunable lipidation mimics. These lipidation mimics can fully recapitulate the biochemical properties of natural lipidation in membrane association for probing its biological functions on signaling proteins and in albumin binding for designing long-acting protein drugs. (2) We demonstrate that the binding affinity between histone methylations and their corresponding readers can be substantially increased with genetically encoded electron-rich Trp derivatives. These engineered affinity-enhanced readers can be applied to enrich, image, and profile the interactome of chromatin methylations. (3) We report the identification and verification of a novel type of protein PTM, aminoacylated lysine ubiquitination, using genetically encoded PTM ncAAs as chemical probes. This approach provides a general strategy for the identification of unknown PTMs by increasing the abundance of PTM bait probes.

Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations.

Many proteoforms can be produced from a gene due to genetic mutations, alternative splicing, post-translational modifications (PTMs), and other variations. PTMs in proteoforms play critical roles in cell signaling, protein degradation, and other biological processes. Mass spectrometry (MS) is the primary technique for investigating PTMs in proteoforms, and two alternative MS approaches, top-down and bottom-up, have complementary strengths. The combination of the two approaches has the potential to increase the sensitivity and accuracy in PTM identification and characterization. In addition, protein and PTM knowledge bases, such as UniProt, provide valuable information for PTM characterization and verification. Here, we present a software pipeline PTM-TBA (PTM characterization by Top-down and Bottom-up MS and Annotations) for identifying and localizing PTMs in proteoforms by integrating top-down and bottom-up MS as well as PTM annotations. We assessed PTM-TBA using a technical triplicate of bottom-up and top-down MS data of SW480 cells. On average, database search of the top-down MS data identified 2000 mass shifts, 814.5 (40.7%) of which were matched to 11 common PTMs and 423 of which were localized. Of the mass shifts identified by top-down MS, PTM-TBA verified 435 mass shifts using the bottom-up MS data and UniProt annotations.

Impact of Bioinformatics Search Parameters for Peptides' Identification and Their Post-Translational Modifications: A Case Study of Proteolysed Gelatines from Beef, Pork, and Fish.

Bioinformatics software, allowing the identification of peptides by the comparison of peptide fragmentation spectra obtained by mass spectrometry versus targeted databases or directly by de novo sequencing, is now mandatory in peptidomics/proteomics approaches. Programming the identification software requires specifying, among other things, the mass measurement accuracy of the instrument and the digestion enzyme used with the number of missed cleavages allowed. Moreover, these software algorithms are able to identify a large number of post-translational modifications (PTMs). However, peptide and PTM identifications are challenging in the agrofood field due to non-specific cleavage sites of physiological- or food-grade enzymes and the number and location of PTMs. In this study, we show the importance of customized software programming to obtain a better peptide and PTM identification rate in the agrofood field. A gelatine product and one industrial gelatine hydrolysate from three different sources (beef, pork, and fish), each digested by simulated gastrointestinal digestion, MS-grade trypsin, or both, were used to perform the comparisons. Two main points are illustrated: (i) the impact of the set-up of specific enzyme versus no specific enzyme use and (ii) the impact of a maximum of six PTMs allowed per peptide versus the standard of three. Prior knowledge of the composition of the raw proteins is an important asset for better identification of peptide sequences.

Elucidating the Implications of Norovirus N- and O-Glycosylation, O-GlcNAcylation, and Phosphorylation.

Norovirus is the most common cause of foodborne gastroenteritis, affecting millions of people worldwide annually. Among the ten genotypes (GI-GX) of norovirus, only GI, GII, GIV, GVIII, and GIX infect humans. Some genotypes reportedly exhibit post-translational modifications (PTMs), including N- and O-glycosylation, O-GlcNAcylation, and phosphorylation, in their viral antigens. PTMs have been linked to increased viral genome replication, viral particle release, and virulence. Owing to breakthroughs in mass spectrometry (MS) technologies, more PTMs have been discovered in recent years and have contributed significantly to preventing and treating infectious diseases. However, the mechanisms by which PTMs act on noroviruses remain poorly understood. In this section, we outline the current knowledge of the three common types of PTM and investigate their impact on norovirus pathogenesis. Moreover, we summarize the strategies and techniques for the identification of PTMs.

Post‐translational modification and phenotype

Post‐translational modification and phenotype

In-depth analysis of the Sirtuin 5-regulated mouse brain malonylome and succinylome using library-free data-independent acquisitions.

Post-translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry-based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data-independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label-free quantification. In this study, we present a powerful approach that combines antibody-based enrichment with comprehensive DIA acquisitions and spectral library-free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, while offering a fully automated workflow. We analyzed brains from wild-type and Sirtuin 5 (SIRT5)-knock-out mice, and discovered and quantified 466 malonylated and 2211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 164 malonylated and 578 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 common SIRT5-regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome.

DIGE Analysis of Clinical Specimens.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an elegant gel electrophoretic analytical tool for comparative protein assessment. It is based on two-dimensional gel electrophoresis (2D-GE) separation of fluorescently labeled protein extracts. The tagging procedures are designed to not interfere with the chemical properties of proteins with respect to their pI and electrophoretic mobility, once a proper labeling protocol is followed. The use of an internal pooled standard makes 2D-DIGE a highly accurate quantitative method enabling multiple protein samples to be separated on the same two-dimensional gel. Technical limitations of this technique (i.e., underrating of low abundant, high molecular mass and integral membrane proteins) are counterbalanced by the incomparable separation power which allows proteoforms and unknown PTM (posttranslational modification) identification. Moreover, the image matching and cross-gel statistical analysis generates robust quantitative results making data validation by independent technologies successful.

Uncommon posttranslational modifications in proteomics: ADP-ribosylation, tyrosine nitration, and tyrosine sulfation.

Posttranslational modifications (PTMs) are covalent modifications of proteins that modulate the structure and functions of proteins and regulate biological processes. The development of various mass spectrometry-based proteomics workflows has facilitated the identification of hundreds of PTMs and aided the understanding of biological significance in a high throughput manner. Improvements in sample preparation and PTM enrichment techniques, instrumentation for liquid chromatography-tandem mass spectrometry (LC-MS/MS), and advanced data analysis tools enhance the specificity and sensitivity of PTM identification. Highly prevalent PTMs like phosphorylation, glycosylation, acetylation, ubiquitinylation, and methylation are extensively studied. However, the functions and impact of less abundant PTMs are not as well understood and underscore the need for analytical methods that aim to characterize these PTMs. This review focuses on the advancement and analytical challenges associated with the characterization of three less common but biologically relevant PTMs, specifically, adenosine diphosphate-ribosylation, tyrosine sulfation, and tyrosine nitration. The advantages and disadvantages of various enrichment, separation, and MS/MS techniques utilized to identify and localize these PTMs are described.

Assessment of label-free quantification and missing value imputation for proteomics in non-human primates

BackgroundReliable and effective label-free quantification (LFQ) analyses are dependent not only on the method of data acquisition in the mass spectrometer, but also on the downstream data processing, including software tools, query database, data normalization and imputation. In non-human primates (NHP), LFQ is challenging because the query databases for NHP are limited since the genomes of these species are not comprehensively annotated. This invariably results in limited discovery of proteins and associated Post Translational Modifications (PTMs) and a higher fraction of missing data points. While identification of fewer proteins and PTMs due to database limitations can negatively impact uncovering important and meaningful biological information, missing data also limits downstream analyses (e.g., multivariate analyses), decreases statistical power, biases statistical inference, and makes biological interpretation of the data more challenging. In this study we attempted to address both issues: first, we used the MetaMorphues proteomics search engine to counter the limits of NHP query databases and maximize the discovery of proteins and associated PTMs, and second, we evaluated different imputation methods for accurate data inference. We used a generic approach for missing data imputation analysis without distinguising the potential source of missing data (either non-assigned m/z or missing values across runs).ResultsUsing the MetaMorpheus proteomics search engine we obtained quantitative data for 1622 proteins and 10,634 peptides including 58 different PTMs (biological, metal and artifacts) across a diverse age range of NHP brain frontal cortex. However, among the 1622 proteins identified, only 293 proteins were quantified across all samples with no missing values, emphasizing the importance of implementing an accurate and statiscaly valid imputation method to fill in missing data. In our imputation analysis we demonstrate that Single Imputation methods that borrow information from correlated proteins such as Generalized Ridge Regression (GRR), Random Forest (RF), local least squares (LLS), and a Bayesian Principal Component Analysis methods (BPCA), are able to estimate missing protein abundance values with great accuracy.ConclusionsOverall, this study offers a detailed comparative analysis of LFQ data generated in NHP and proposes strategies for improved LFQ in NHP proteomics data.

Comprehensive Mapping and Dynamics of Site-Specific Prolyl-Hydroxylation, Lysyl-Hydroxylation and Lysyl O-Glycosylation of Collagens Deposited in ECM During Zebrafish Heart Regeneration.

Cardiac fibrosis-mediated heart failure (HF) is one of the major forms of end-stage cardiovascular diseases (CVDs). Cardiac fibrosis is an adaptive response of the myocardium upon any insult/injury. Excessive deposition of collagen molecules in the extracellular matrix (ECM) is the hallmark of fibrosis. This fibrotic response initially protects the myocardium from ventricular rupture. Although in mammals this fibrotic response progresses towards scar-tissue formation leading to HF, some fishes and urodeles have mastered the art of cardiac regeneration following injury-mediated fibrotic response. Zebrafish have a unique capability to regenerate the myocardium after post-amputation injury. Following post-amputation, the ECM of the zebrafish heart undergoes extensive remodeling and deposition of collagen. Being the most abundant protein of ECM, collagen plays important role in the assembly and cell-matrix interactions. However, the mechanism of ECM remodeling is not well understood. Collagen molecules undergo heavy post-translational modifications (PTMs) mainly hydroxylation of proline, lysine, and glycosylation of lysine during biosynthesis. The critical roles of these PTMs are emerging in several diseases, embryonic development, cell behavior regulation, and cell-matrix interactions. The site-specific identification of these collagen PTMs in zebrafish heart ECM is not known. As these highly modified peptides are not amenable to mass spectrometry (MS), the site-specific identification of these collagen PTMs is challenging. Here, we have implemented our in-house proteomics analytical pipeline to analyze two ECM proteomics datasets (PXD011627, PXD010092) of the zebrafish heart during regeneration (post-amputation). We report the first comprehensive site-specific collagen PTM map of zebrafish heart ECM. We have identified a total of 36 collagen chains (19 are reported for the first time here) harboring a total of 95 prolyl-3-hydroxylation, 108 hydroxylysine, 29 galactosyl-hydroxylysine, and 128 glucosylgalactosyl-hydroxylysine sites. Furthermore, we comprehensively map the three chains (COL1A1a, COL1A1b, and COL1A2) of collagen I, the most abundant protein in zebrafish heart ECM. We achieved more than 95% sequence coverage for all the three chains of collagen I. Our analysis also revealed the dynamics of prolyl-3-hydroxylation occupancy oscillations during heart regeneration at these sites. Moreover, quantitative site-specific analysis of lysine-O-glycosylation microheterogeneity during heart regeneration revealed a significant (p < 0.05) elevation of site-specific (K1017) glucosylgalactosyl-hydroxylysine on the col1a1a chain. Taken together, these site-specific PTM maps and the dynamic changes of site-specific collagen PTMs in ECM during heart regeneration will open up new avenues to decode ECM remodeling and may lay the foundation to tinker the cardiac regeneration process with new approaches.

Unique Posttranslational Modification Sites of Acetylation, Citrullination, Glutarylation, and Phosphorylation Are Found to Be Specific to the Proteins Partitioned in the Triton X-114 Fractions of Leptospira.

Posttranslational modifications (PTMs) are decisive factors in the structure, function, and localization of proteins in prokaryotic and eukaryotic organisms. However, prokaryotic organisms lack subcellular organelles, and protein localization based on subcellular locations like cytoplasm, inner membrane, periplasm, and outer membrane can be accounted for functional characterization. We have identified 131 acetylated, 1182 citrullinated, 72 glutarylated, 5 palmitoylated, and 139 phosphorylated proteins from Triton X-114 fractionated proteins of Leptospira, the pathogen of re-emerging zoonotic disease leptospirosis. In total, 74.7% of proteins were found exclusively in different Triton X-114 fractions. Additionally, 21.9% of proteins in multiple fractions had one or more PTM specific to different Triton X-114 fractions. Altogether, 96.6% of proteins showed exclusiveness to different Triton X-114 fractions either due to the presence of the entire protein or with a specific PTM type or position. Further, the PTM distribution within Triton X-114 fractions showed higher acetylation in aqueous, glutarylation in detergent, phosphorylation in pellet, and citrullination in wash fractions representing cytoplasmic, outer membrane, inner membrane, and extracellular locations, respectively. Identification of PTMs in proteins with respect to the subcellular localization will help to characterize candidate proteins before developing novel drugs and vaccines rationally to combat leptospirosis.

Novel Post-translational Modifications in Human Serum Albumin.

This study aims to identify novel post-translational modifications in human serum albumin by mass spectrometry. Serum albumin is the most abundant protein in plasma, has many physiological functions, and is in contact with most of the cells and tissues of the human body. Post-translational modifications (PTMs) may affect functions, stability, and localization of albumin. Human serum albumin (HSA) was used for tryptic digestion in-solution or in-gel. Mass spectrometry was applied to identify PTMs in HSA. 3-dimensional modeling was applied to explore the potential impact of PTMs on known functions of albumin. Here, we report the identification of 61 novel PTMs of human serum albumin. Phosphorylation, glycosylation, nitrosylation, deamidation, methylation, acetylation, palmitoylation, geranylation, and farnesylation are some examples of the identified PTMs. Mass spectrometry was used for the identification of PTMs in a purified HSA and HSA from the human plasma. Threedimensional modeling of albumin with selected PTMs showed the location of these PTMs in the regions involved in albumin interactions with drugs, metals, and fatty acids. The location of PTMs in these regions may modify the binding capacity of albumin. This report adds 61 novel PTMs to the catalog of human albumin.

Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research.

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.

MS-Decipher: a user-friendly proteome database search software with an emphasis on deciphering the spectra of O-linked glycopeptides.

The interpretation of mass spectrometry (MS) data is a crucial step in proteomics analysis, and the identification of post-translational modifications (PTMs) is vital for the understanding of the regulation mechanism of the living system. Among various PTMs, glycosylation is one of the most diverse ones. Though many search engines have been developed to decipher proteomic data, some of them are difficult to operate and have poor performance on glycoproteomic datasets compared to advanced glycoproteomic software. To simplify the analysis of proteomic datasets, especially O-glycoproteomic datasets, here, we present a user-friendly proteomic database search platform, MS-Decipher, for the identification of peptides from MS data. Two scoring schemes can be chosen for peptide-spectra matching. It was found that MS-Decipher had the same sensitivity and confidence in peptide identification compared to traditional database searching software. In addition, a special search mode, O-Search, is integrated into MS-Decipher to identify O-glycopeptides for O-glycoproteomic analysis. Compared with Mascot, MetaMorpheus and MSFragger, MS-Decipher can obtain about 139.9%, 48.8% and 6.9% more O-glycopeptide-spectrum matches. A useful tool is provided in MS-Decipher for the visualization of O-glycopeptide-spectra matches. MS-Decipher has a user-friendly graphical user interface, making it easier to operate. Several file formats are available in the searching and validation steps. MS-Decipher is implemented with Java, and can be used cross-platform. MS-Decipher is freely available at https://github.com/DICP-1809/MS-Decipher for academic use. For detailed implementation steps, please see the user guide. Supplementary data are available at Bioinformatics online.

Nuclei Enrichment from Sugarcane Stems for Proteomics Analyses.

Nuclei enrichment procedures enable a large variety of investigations. These studies include structural characterization of nuclear proteins, identification of posttranslational modifications, and regulation of stress or development-related gene expression. Successful enrichment of nuclei samples from plant tissues is crucial for a comprehensive analysis of the plant nuclear proteome. Here, we describe a method for nuclei enrichment from sugarcane stems and its assessment by western blot.

Post-translational Modifications in Oral Bacteria and Their Functional Impact

Oral bacteria colonize the oral cavity, surrounding complex and variable environments. Post-translational modifications (PTMs) are an efficient biochemical mechanism across all domains of life. Oral bacteria could depend on PTMs to quickly regulate their metabolic processes in the face of external stimuli. In recent years, thanks to advances in enrichment strategies, the number and variety of PTMs that have been identified and characterized in oral bacteria have increased. PTMs, covalently modified by diverse enzymes, occur in amino acid residues of the target substrate, altering the functions of proteins involved in different biological processes. For example, Ptk1 reciprocally phosphorylates Php1 on tyrosine residues 159 and 161, required for Porphyromonas gingivalis EPS production and community development with the antecedent oral biofilm constituent Streptococcus gordonii, and in turn Php1 dephosphorylates Ptk1 and rapidly causes the conversion of Ptk1 to a state of low tyrosine phosphorylation. Protein acetylation is also widespread in oral bacteria. In the acetylome of Streptococcus mutans, 973 acetylation sites were identified in 445 proteins, accounting for 22.7% of overall proteins involving virulence factors and pathogenic processes. Other PTMs in oral bacteria include serine or threonine glycosylation in Cnm involving intracerebral hemorrhage, arginine citrullination in peptidylarginine deiminases (PADs), leading to inflammation, lysine succinylation in P. gingivalis virulence factors (gingipains, fimbriae, RagB, and PorR), and cysteine glutathionylation in thioredoxin-like protein (Tlp) in response to oxidative stress in S. mutans. Here we review oral bacterial PTMs, focusing on acetylation, phosphorylation, glycosylation, citrullination, succinylation, and glutathionylation, and corresponding modifying enzymes. We describe different PTMs in association with some examples, discussing their potential role and function in oral bacteria physiological processes and regulatory networks. Identification and characterization of PTMs not only contribute to understanding their role in oral bacterial virulence, adaption, and resistance but will open new avenues to treat oral infectious diseases.

Deconvoluting complex protein interaction networks through reductionist strategies in peptide biochemistry: Modern approaches and research questions.

Following the decoding of the first human genome, researchers have vastly improved their understanding of cell biology and its regulation. As a result, it has become clear that it is not merely genetic information, but the aberrant changes in the functionality and connectivity of its encoded proteins that drive cell response to periods of stress and external cues. Therefore, proper utilization of refined methods that help to describe protein signalling or regulatory networks (i.e., functional connectivity), can help us understand how change in the signalling landscape effects the cell. However, given the vast complexity in 'how and when' proteins communicate or interact with each other, it is extremely difficult to define, characterize, and understand these interaction networks in a tangible manner. Herein lies the challenge of tackling the functional proteome; its regulation is encoded in multiple layers of interaction, chemical modification and cell compartmentalization. To address and refine simple research questions, modern reductionist strategies in protein biochemistry have successfully used peptide-based experiments; their summation helping to simplify the overall complexity of these protein interaction networks. In this way, peptides are powerful tools used in fundamental research that can be readily applied to comparative biochemical research. Understanding and defining how proteins interact is one of the key aspects towards understanding how the proteome functions. To date, reductionist peptide-based research has helped to address a wide range of proteome-related research questions, including the prediction of enzymes substrates, identification of posttranslational modifications, and the annotation of protein interaction partners. Peptide arrays have been used to identify the binding specificity of reader domains, which are able to recognise the posttranslational modifications; forming dynamic protein interactions that are dependent on modification state. Finally, representing one of the fastest growing classes of inhibitor molecules, peptides are now begin explored as "disruptors" of protein-protein interactions or enzyme activity. Collectively, this review will discuss the use of peptides, peptide arrays, peptide-oriented computational biochemistry as modern reductionist strategies in deconvoluting the functional proteome.