scholarly journals Clinically Relevant Post-Translational Modification Analyses—Maturing Workflows and Bioinformatics Tools

2018 ◽  
Vol 20 (1) ◽  
pp. 16 ◽  
Author(s):  
Dana Pascovici ◽  
Jemma X. Wu ◽  
Matthew J. McKay ◽  
Chitra Joseph ◽  
Zainab Noor ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Data Interpretation ◽  
Label Free ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Data Independent Acquisition ◽  
Bioinformatics Tools ◽  
Functional Understanding ◽  
Health And Disease ◽  
Regulate Protein

Post-translational modifications (PTMs) can occur soon after translation or at any stage in the lifecycle of a given protein, and they may help regulate protein folding, stability, cellular localisation, activity, or the interactions proteins have with other proteins or biomolecular species. PTMs are crucial to our functional understanding of biology, and new quantitative mass spectrometry (MS) and bioinformatics workflows are maturing both in labelled multiplexed and label-free techniques, offering increasing coverage and new opportunities to study human health and disease. Techniques such as Data Independent Acquisition (DIA) are emerging as promising approaches due to their re-mining capability. Many bioinformatics tools have been developed to support the analysis of PTMs by mass spectrometry, from prediction and identifying PTM site assignment, open searches enabling better mining of unassigned mass spectra—many of which likely harbour PTMs—through to understanding PTM associations and interactions. The remaining challenge lies in extracting functional information from clinically relevant PTM studies. This review focuses on canvassing the options and progress of PTM analysis for large quantitative studies, from choosing the platform, through to data analysis, with an emphasis on clinically relevant samples such as plasma and other body fluids, and well-established tools and options for data interpretation.

scholarly journals Quantification and Identification of Post-Translational Modifications Using Modern Proteomics

2021 ◽  
pp. 225-235
Author(s):  
Anja Holtz ◽  
Nathan Basisty ◽  
Birgit Schilling
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
High Throughput ◽  
High Resolution Mass Spectrometry ◽  
Label Free ◽  
Post Translational Modifications ◽  
Data Independent Acquisition ◽  
Lysine Residues ◽  
Detailed Protocol ◽  
Resolution Mass

AbstractPost-translational modifications (PTMs) occur dynamically, allowing cells to quickly respond to changes in the environment. Lysine residues can be targeted by several modifications including acylations (acetylation, succinylation, malonylation, glutarylation, and others), methylation, ubiquitination, and other modifications. One of the most efficient methods for the identification of post-translational modifications is utilizing immunoaffinity enrichment followed by high-resolution mass spectrometry. This workflow can be coupled with comprehensive data-independent acquisition (DIA) mass spectrometry to be a high-throughput, label-free PTM quantification approach. Below we describe a detailed protocol to process tissue by homogenization and proteolytically digest proteins, followed by immunoaffinity enrichment of lysine-acetylated peptides to identify and quantify relative changes of acetylation comparing different conditions.

Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

2020 ◽  
Vol 64 (1) ◽  
pp. 97-110
Author(s):  
Christian Sibbersen ◽  
Mogens Johannsen
Keyword(s):  
Mass Spectrometry ◽  
Future Research ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Dicarbonyl Compounds ◽  
Protein Targets ◽  
Post Translational Modifications ◽  
Reactive Protein ◽  
Glycation End Products ◽  
Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

scholarly journals Emerging mass spectrometry-based proteomics methodologies for novel biomedical applications

2020 ◽  
Vol 48 (5) ◽  
pp. 1953-1966
Author(s):  
Lindsay K. Pino ◽  
Jacob Rose ◽  
Amy O'Broin ◽  
Samah Shah ◽  
Birgit Schilling
Keyword(s):  
Mass Spectrometry ◽  
Human Health ◽  
Protein Complexes ◽  
Protein Quantification ◽  
Intact Protein ◽  
Protein Signaling ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Health And Disease ◽  
Proteomics Research

Research into the basic biology of human health and disease, as well as translational human research and clinical applications, all benefit from the growing accessibility and versatility of mass spectrometry (MS)-based proteomics. Although once limited in throughput and sensitivity, proteomic studies have quickly grown in scope and scale over the last decade due to significant advances in instrumentation, computational approaches, and bio-sample preparation. Here, we review these latest developments in MS and highlight how these techniques are used to study the mechanisms, diagnosis, and treatment of human diseases. We first describe recent groundbreaking technological advancements for MS-based proteomics, including novel data acquisition techniques and protein quantification approaches. Next, we describe innovations that enable the unprecedented depth of coverage in protein signaling and spatiotemporal protein distributions, including studies of post-translational modifications, protein turnover, and single-cell proteomics. Finally, we explore new workflows to investigate protein complexes and structures, and we present new approaches for protein–protein interaction studies and intact protein or top-down MS. While these approaches are only recently incipient, we anticipate that their use in biomedical MS proteomics research will offer actionable discoveries for the improvement of human health.

scholarly journals Selection of Features with Consistent Profiles Improves Relative Protein Quantification in Mass Spectrometry Experiments

2020 ◽  
Vol 19 (6) ◽  
pp. 944-959 ◽  
Author(s):  
Tsung-Heng Tsai ◽  
Meena Choi ◽  
Balazs Banfai ◽  
Yansheng Liu ◽  
Brendan X. MacLean ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Profile ◽  
Protein Quantification ◽  
Selected Reaction Monitoring ◽  
Estimation Accuracy ◽  
Spectral Features ◽  
Data Set ◽  
Post Translational Modifications ◽  
Data Independent Acquisition ◽  
Manual Curation

In bottom-up mass spectrometry-based proteomics, relative protein quantification is often achieved with data-dependent acquisition (DDA), data-independent acquisition (DIA), or selected reaction monitoring (SRM). These workflows quantify proteins by summarizing the abundances of all the spectral features of the protein (e.g. precursor ions, transitions or fragments) in a single value per protein per run. When abundances of some features are inconsistent with the overall protein profile (for technological reasons such as interferences, or for biological reasons such as post-translational modifications), the protein-level summaries and the downstream conclusions are undermined. We propose a statistical approach that automatically detects spectral features with such inconsistent patterns. The detected features can be separately investigated, and if necessary, removed from the data set. We evaluated the proposed approach on a series of benchmark-controlled mixtures and biological investigations with DDA, DIA and SRM data acquisitions. The results demonstrated that it could facilitate and complement manual curation of the data. Moreover, it can improve the estimation accuracy, sensitivity and specificity of detecting differentially abundant proteins, and reproducibility of conclusions across different data processing tools. The approach is implemented as an option in the open-source R-based software MSstats.

scholarly journals NAguideR: performing and prioritizing missing value imputations for consistent bottom-up proteomic analyses

2020 ◽  
Vol 48 (14) ◽  
pp. e83-e83 ◽  
Author(s):  
Shisheng Wang ◽  
Wenxue Li ◽  
Liqiang Hu ◽  
Jingqiu Cheng ◽  
Hao Yang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Missing Values ◽  
Protein Complexes ◽  
Label Free ◽  
Missing Value ◽  
Imputation Methods ◽  
Data Independent Acquisition ◽  
Low Performance ◽  
User Friendly

Abstract Mass spectrometry (MS)-based quantitative proteomics experiments frequently generate data with missing values, which may profoundly affect downstream analyses. A wide variety of imputation methods have been established to deal with the missing-value issue. To date, however, there is a scarcity of efficient, systematic, and easy-to-handle tools that are tailored for proteomics community. Herein, we developed a user-friendly and powerful stand-alone software, NAguideR, to enable implementation and evaluation of different missing value methods offered by 23 widely used missing-value imputation algorithms. NAguideR further evaluates data imputation results through classic computational criteria and, unprecedentedly, proteomic empirical criteria, such as quantitative consistency between different charge-states of the same peptide, different peptides belonging to the same proteins, and individual proteins participating protein complexes and functional interactions. We applied NAguideR into three label-free proteomic datasets featuring peptide-level, protein-level, and phosphoproteomic variables respectively, all generated by data independent acquisition mass spectrometry (DIA-MS) with substantial biological replicates. The results indicate that NAguideR is able to discriminate the optimal imputation methods that are facilitating DIA-MS experiments over those sub-optimal and low-performance algorithms. NAguideR further provides downloadable tables and figures supporting flexible data analysis and interpretation. NAguideR is freely available at http://www.omicsolution.org/wukong/NAguideR/ and the source code: https://github.com/wangshisheng/NAguideR/.

Label-Free Quantification by Data Independent Acquisition Mass Spectrometry to Map Cardiovascular Proteomes

2016 ◽  
pp. 227-245
Author(s):  
Sarah J. Parker ◽  
Ronald J. Holewinski ◽  
Irina Tchernyshyov ◽  
Vidya Venkatraman ◽  
Laurie Parker ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Label Free ◽  
Label Free Quantification ◽  
Data Independent Acquisition ◽  
Free Quantification

scholarly journals FLEXIQuant-LF to quantify protein modification extent in label-free proteomics data

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Christoph N Schlaffner ◽  
Konstantin Kahnert ◽  
Jan Muntel ◽  
Ruchi Chauhan ◽  
Bernhard Y Renard ◽  
...  
Keyword(s):  
Protein Modification ◽  
Large Scale ◽  
Software Tool ◽  
Label Free ◽  
Anaphase Promoting Complex ◽  
Proteomics Data ◽  
Single Experiment ◽  
Post Translational Modifications ◽  
Data Independent Acquisition ◽  
Modified Peptides

Improvements in LC-MS/MS methods and technology have enabled the identification of thousands of modified peptides in a single experiment. However, protein regulation by post-translational modifications (PTMs) is not binary, making methods to quantify the modification extent crucial to understanding the role of PTMs. Here, we introduce FLEXIQuant-LF, a software tool for large-scale identification of differentially modified peptides and quantification of their modification extent without knowledge of the types of modifications involved. We developed FLEXIQuant-LF using label-free quantification of unmodified peptides and robust linear regression to quantify the modification extent of peptides. As proof of concept, we applied FLEXIQuant-LF to data-independent-acquisition (DIA) data of the anaphase promoting complex/cyclosome (APC/C) during mitosis. The unbiased FLEXIQuant-LF approach to assess the modification extent in quantitative proteomics data provides a better understanding of the function and regulation of PTMs. The software is available at https://github.com/SteenOmicsLab/FLEXIQuantLF.

Analysis of the uterine flush fluid proteome of healthy mares and mares with endometritis or fibrotic endometrial degeneration

2020 ◽  
Vol 32 (6) ◽  
pp. 572 ◽  
Author(s):  
Mariana Diel de Amorim ◽  
Firdous A. Khan ◽  
Tracey S. Chenier ◽  
Elizabeth L. Scholtz ◽  
M. Anthony Hayes
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass ◽  
Label Free ◽  
Chromatography Tandem Mass Spectrometry ◽  
Uterine Health ◽  
Liquid Chromatography Tandem Mass ◽  
Specific Proteins ◽  
Health And Disease ◽  
Flush Fluid

The objective of this study was to evaluate the differences in the uterine flush fluid proteome between healthy mares and mares with endometritis or fibrotic endometrial degeneration (FED). Uterine flush fluid samples were collected from healthy mares (n=8; oestrus n=5 and dioestrus n=3) and mares with endometritis (n=23; oestrus n=14 and dioestrus n=9) or FED (n=7; oestrus n=6 and dioestrus n=1). Proteomic analysis was performed using label-free liquid chromatography–tandem mass spectrometry. Of 216 proteins identified during oestrus, 127 were common to all three groups, one protein was exclusively detected in healthy mares, 47 proteins were exclusively detected in mares with endometritis and four proteins were exclusively detected in mares with FED. Of 188 proteins identified during dioestrus, 113 proteins were common between healthy mares and mares with endometritis, eight proteins were exclusively detected in healthy mares and 67 proteins were exclusively detected in mares with endometritis. Quantitative analysis revealed a subset of proteins differing in abundance between the three groups during oestrus and between healthy mares and mares with endometritis during dioestrus. These results provide a springboard for evaluation of specific proteins as biomarkers of uterine health and disease and for investigation of their roles in the establishment and maintenance of pregnancy.

Abstract 355: Post-translational Modification of Histone Variants in Cardiac Hypertrophy

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Mickey Miller ◽  
Aman Makaju ◽  
Li Wang ◽  
Sarah Franklin
Keyword(s):  
Gene Expression ◽  
Cardiac Hypertrophy ◽  
Differential Expression Analysis ◽  
Pressure Overload ◽  
Histone Variants ◽  
Neonatal Rat ◽  
Global Changes ◽  
Label Free ◽  
Post Translational Modification ◽  
Post Translational Modifications

While global changes in gene expression are a hallmark of cardiac hypertrophy, much less is known regarding the epigenetic factors driving these changes. Local chromatin packing and gene accessibility, which governs transcriptional status, has been correlated with specific post-translational modifications on the histone tails of nucleosomes occupying these regions. However, the specific alterations in histone post-translational modifications driving gene expression changes during cardiac hypertrophy are largely unknown. To identify myocyte specific changes in histone post-translational modifications during cardiac hypertrophy we performed label-free quantitation of nuclear proteins from isolated neonatal rat ventricular myocytes exposed to the hypertrophic agonists, phenylephrine and isoproterenol. Peptide samples were analyzed on a Thermo Orbitrap Velos Pro mass spectrometer using CID & HCD fragmentation. Differential expression analysis was performed using the Progenesis LC-MS software where modified histone peptides were normalized against total protein expression. We observed multiple known and novel post-translational modifications on each of the four core histones, many of which changed in the setting of hypertrophy. To validate these findings in an animal model we performed the same analysis of histone post-translational modifications from cardiac tissue of mice under basal conditions or after pressure-overload induced hypertrophy. This study provides the first global characterization of myocyte specific changes in histone post-translational modifications in cardiac hypertrophy and highlight basic mechanisms of genomic reprogramming operative in disease.

scholarly journals In-Depth Mapping of the Urinary N-Glycoproteome: Distinct Signatures of ccRCC-related Progression

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 239 ◽  
Author(s):  
Lucia Santorelli ◽  
Giulia Capitoli ◽  
Clizia Chinello ◽  
Isabella Piga ◽  
Francesca Clerici ◽  
...  
Keyword(s):  
Clear Cell ◽  
Glycosylation Site ◽  
Biological Processes ◽  
Label Free ◽  
Post Translational Modification ◽  
Cell Renal Cell Carcinoma ◽  
Altered Expression ◽  
Post Translational Modifications ◽  
Novel Biomarkers

Protein N-glycosylation is one of the most important post-translational modifications and is involved in many biological processes, with aberrant changes in protein N-glycosylation patterns being closely associated with several diseases, including the progression and spreading of tumours. In light of this, identifying these aberrant protein glycoforms in tumours could be useful for understanding the molecular mechanism of this multifactorial disease, developing specific biomarkers and finding novel therapeutic targets. We investigated the urinary N-glycoproteome of clear cell renal cell carcinoma (ccRCC) patients at different stages (n = 15 at pT1 and n = 15 at pT3), and of non-ccRCC subjects (n = 15), using an N-glyco-FASP-based method. Using label-free nLC-ESI MS/MS, we identified and quantified several N-glycoproteins with altered expression and abnormal changes affecting the occupancy of the glycosylation site in the urine of RCC patients compared to control. In particular, nine of them had a specific trend that was directly related to the stage progression: CD97, COCH and P3IP1 were up-expressed whilst APOB, FINC, CERU, CFAH, HPT and PLTP were down-expressed in ccRCC patients. Overall, these results expand our knowledge related to the role of this post-translational modification in ccRCC and translation of this information into pre-clinical studies could have a significant impact on the discovery of novel biomarkers and therapeutic target in kidney cancer.

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