Search

High-throughput quantitative analysis of the cells' proteomes across multiple conditions such as various perturbations and different time points is essential for gaining insights into treatment-induced biological responses or disease pathological states. The advancements in mass spectrometry instrumentation and isobaric labeling methods provided useful tools to help address such demands. However, the current widely adopted isobaric labeling methods such as tandem mass tag (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ) are based on low-mass reporter ions, which are indistinguishable among different peptide analytes, to achieve relative quantification. Therefore, these methods intrinsically suffer from severe ratio distortion when analyzing complex samples due to peptide coelution and cofragmentation. Here, we developed a novel set of isobaric tags named dimethylated leucine complementary ion (DiLeuC) and relied on complementary ions for relative quantification, in which the complementary ions are the remanent peptide segments after fragmentation in the high-mass range. Since those residual peptide fragments are precursor-specific, they retain the relative abundance information in an interference-free manner even in a complex matrix environment. The quantification accuracy of our method was validated in a two-proteome model where the yeast proteome was spiked with a strong background human proteome as interference. In addition, we also applied this strategy to single-cell proteome analysis, demonstrating its potential utility for sensitive high-throughput quantitative proteomics. [ABSTRACT FROM AUTHOR]

The quantitative multiplexing capacity of isobaric tandem mass tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated bait proteins. However, variable bait levels between replicates can convolute interactor identification. We compared the Student's t-test and Pearson's R correlation as methods to generate t-statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly controlling type I errors (false positives). We experimentally determined the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8H31Q followed by stringent washing, and reversible association to 14-3-3ζ with gentle washing. Correlation-derived t-statistics performed at least as well as Student's t-statistics for each sample and with substantial improvement in performance for experiments with high bait-level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase the robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors using TMT-AP-MS. Data are available via ProteomeXchange with identifier PXD016613.

The standard approach for proteomic data acquisition of isobaric-tagged samples by mass spectrometry is data-dependent acquisition. This semistochastic, identification-first paradigm generates a wealth of peptide-level data without regard to relative abundance. We introduce a data acquisition concept called sequential windowed acquisition of reporter masses (SWARM). This approach performs quantitation first, thereby allowing subsequent acquisition decisions to be predicated on user-defined patterns of reporter ion intensities. The efficacy of this approach is validated through experiments with both synthetic mixtures of Escherichia coli ribosomes spiked into human cell lysates at known ratios and the quantitative evaluation of the human proteome's response to the inhibition of cullin-based protein ubiquitination via the small molecule MLN4924. We find that SWARM-informed parallel reaction monitoring acquisitions display effective acquisition biasing toward analytes displaying quantitative characteristics of interest, resulting in an improvement in the detection of differentially abundant analytes. The SWARM concept provides a flexible platform for the further development of new acquisition methods.

This review explores the diverse applications of deuterium in medicinal chemistry, particularly as an analytical tool in mass spectrometry. Deuterium applications extend beyond its traditional use as an analytical reference material to being integrated into drug structures and tagging reagents for quantitative proteomics. Hydrogen-deuterium exchange (HDX) of labile protons is applied for structural analysis and monitoring protein folding, interactions, and denaturation processes, whereas the metal-catalyzed HDX in C–H bonds provides site-specific deuterium labeling. Deuterated analytical reference materials and labeling reagents are used in the investigation of metabolic pathways, offering valuable insights into drug transformations. Deuterium has evolved into a powerful tool for mass spectrometry-based structural and quantitative analyses, however, challenges such as isotopic effects, chromatographic elution profile differences, and proton mixing in MS analysis still exist, necessitating ongoing technological advancements. [Display omitted] • Deuterium-labeled compounds are synthesized from scratch or obtained through HDX. • Labile proton exchange provides insightful structural information in proteomics. • Deuterated drugs are being approved due to enhanced stability and therapeutic efficacy. • HDX effects are studied by high resolution mass spectrometry. • Deuterium labeled compounds are routinely used in quantitative and qualitative omics. [ABSTRACT FROM AUTHOR]

STIM1 is an ER/SR transmembrane protein that interacts with ORAI1 to activate store operated Ca2+ entry (SOCE) upon ER/SR depletion of calcium. Normally highly expressed in skeletal muscle, STIM1 deficiency causes significant changes to mitochondrial ultrastructure that do not occur with loss of ORAI1 or other components of SOCE. The datasets in this article are from large-scale proteomics and phosphoproteomics experiments in an inducible mouse model of skeletal muscle-specific STIM1 knock out (KO). These data reveal statistically significant changes in the relative abundance of specific proteins and sites of protein phosphorylation in STIM1 KO gastrocnemius. Protein samples from five biological replicates of each condition (+/- STIM1) were enzymatically digested, the resulting peptides labeled with tandem mass tag (TMT) reagents, mixed, and fractionated. Phosphopeptides were enriched and a small amount of each input retained for protein abundance analysis. All phosphopeptide and input fractions were analyzed by nano LC-MS/MS on a Q Exactive Plus Orbitrap mass spectrometer, searched with Proteome Discoverer software, and processed with in-house R-scripts for data normalization and statistical analysis. Article published in Molecular Metabolism [1].

The objective of the study was to examine the variations in sarcoplasmic proteomes of bison longissimus lumborum (LL) and psoas major (PM) muscles during postmortem aging utilizing tandem mass tag isobaric labeling coupled with liquid chromatography–mass spectrometry for the categorization of muscles with muscle-specific inherent color stability. A total of 576 proteins were identified in both bison LL and PM muscles, where 97 proteins were identified as differentially abundant (fold change > 1.5, P < 0.05) from the 3 comparisons between muscles during postmortem aging periods (PM vs. LL at 2 d, 7 d, and 14 d). Between muscles, the most abundant protein groups were based on functions such as electron transport chain or oxidative phosphorylation, tricarboxylic acid cycle, adenosine triphosphate transport, carbohydrate metabolism, fatty acid oxidation, chaperones, oxygen transport, muscle contraction, calcium signaling, and protein synthesis. In PM, most of the proteins from electron transport chain, tricarboxylic acid cycle, fatty acid oxidation, adenosine triphosphate and oxygen transport, and muscle contraction were more abundant or exhibited increased expression during aging compared with LL. On the other hand, the proteins involved in carbohydrate metabolism, chaperone function, and protein synthesis mostly exhibited decreased expression in PM muscles relative to LL. These results clearly demonstrate that the proteins associated with oxidative metabolism showed increased expression in PM muscles. This indicates that oxidative damage and subsequent color deterioration resulted in bison PM muscles being attacked by the reactive oxygen species produced during those metabolic processes. In contrast, proteins involved in glycolysis and chaperone activity exhibited a decrease in expression in bison PM muscles, resulting in a decline in color stability compared with LL. Because glycolytic enzymes generate reducing equivalents and chaperones maintain the native folded protein structure, they are consequently responsible for the color stability in LL muscles compared with PM.

Peptidomics is the detection and identification of the peptides present in a sample, and quantitative peptidomics provides additional information about the amounts of these peptides. It is possible to perform absolute quantitation of peptide levels in which the biological sample is compared to synthetic standards of each peptide. More commonly, relative quantitation is performed to compare peptide levels between two or more samples. Relative quantitation can measure differences between all peptides that are detectable, which can exceed 1000 peptides in a complex sample. In this chapter, various techniques used for quantitative peptidomics are described along with discussion of the advantages and disadvantages of each approach. A guide to selecting the optimal quantitative approach is provided, based on the goals of the experiment and the resources that are available., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The objective of this study was to identify the S-nitrosylated proteins in aging samples of pork longissimus thoracis muscle (aged 0 and 3 d) and to study the effects of exogenous S-nitrosoglutathione (GSNO, concentration at 10 and 100 μM) treatments of aged 0 d sample. After validating modified biotin switch method, the samples were labeled with tandem mass tags (TMT126-129) for the LC-MS/MS analysis. A total of 366 peptides were identified to be S-nitrosylated corresponding to 339 proteins. Comparison of total intensity and individual S-nitrosylated sites between aging samples revealed that S-nitrosylation did occur in pork muscle during postmortem aging through possible pathways of denitrosylation and transnitrosylation. GSNO treatment groups showed a considerable number of potential cysteines could be modified with high thiol-reactivity. It was deduced that S-nitrosylation could be involved in the postmortem metabolic process possibly through the regulation of activity or function of glycolytic enzymes, calcium release, heat shock proteins, antioxidant enzymes and myofibrillar proteins. [ABSTRACT FROM AUTHOR]

An optimized workflow for multiplexed and spatially localized on-tissue quantitative protein analysis is here presented. The method is based on the use of an enzyme delivery platform, a polymeric hydrogel disc, allowing for a localized digestion directly onto the tissue surface coupled with an isobaric mass tag strategy for peptide labeling and relative quantification. The digestion occurs within such hydrogels, followed by peptide solvent extraction and identification by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Since this is a histology-directed on-tissue analysis, multiple hydrogels were placed onto morphologically and spatially different regions of interest (ROIs) within the tissue surface, e.g., cardiac myxoma tumor vascularized region and the adjacent hypocellular area. After a microwave digestion step (2 min), enzymatically cleaved peptides were labeled using TMT reagents with isobaric mass tags, enabling analysis of multiple samples per experiment. Thus, N = 8 hydrogel-digested samples from cardiac myxoma serial tissue sections ( N = 4 from the vascularized ROIs and N = 4 from the adjacent hypocellular areas) were processed and then combined before a single LC-MS/MS analysis. Regulated proteins from both cardiac myxoma regions were assayed in a single experiment. [ABSTRACT FROM AUTHOR]

Glycans, which are widely distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as immune response and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probes greatly facilitates routine analysis of glycans. However, the requirement of high-throughput analysis still calls for advanced tools to be developed. Recently, we devised i s obaric m u ltiplex rea g ents for c ar bonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the subtle mass differences among different isotopologues and expanded the multiplexing capacity to 12 channels, a 3-fold throughput improvement for the original SUGAR tag design and achieved high-throughput N-glycan analysis in a single LC-MS/MS injection. We then applied 12-plex SUGAR tags to profile the N-glycans in four subtypes of human Immunoglobulin G (IgG) and to investigate the N-glycan changes in the endometrial cancer cells (ECC1) treated with Atovaquone, a quinone antimicrobial medication, and a dihydroorotate dehydrogenase (DHODH) inhibitor. Data are available via ProteomeXchange with the identifier PXD038501.

Despite many years of studies, ovarian cancer remains one of the top ten cancers worldwide. Its high mortality rate is mainly due to lack of sufficient diagnostic methods. For this reason, our research focused on the identification of blood markers whose appearance would precede the clinical manifestation of the disease. ITRAQ-tagging (isobaric Tags for Relative and Absolute Quantification) coupled with mass spectrometry technology was applied. Three groups of samples derived from patients with: ovarian cancer, benign ovarian tumor, and healthy controls, were examined. Mass spectrometry analysis allowed for highlighting the dysregulation of several proteins associated with ovarian cancer. Further validation of the obtained results indicated that five proteins (Serotransferrin, Amyloid A1, Hemopexin, C-reactive protein, Albumin) were differentially expressed in ovarian cancer group. Interestingly, the addition of Albumin, Serotransferrin, and Amyloid A1 to CA125 (cancer antigen 125) and HE4 (human epididymis protein4) improved the diagnostic performance of the model discriminating between benign and malignant tumors. Identified proteins shed light on the molecular signaling pathways that are associated with ovarian cancer development and should be further investigated in future studies. Our findings indicate five proteins with a strong potential to use in a multimarker test for screening and detection of ovarian cancer.

This study aimed to investigate the molecular mechanisms underlying the antibiotic resistance difference among three Acinetobacter baumannii isolates. Fifty A. baumannii isolates were first subjected to an antimicrobial susceptibility test, then three isolates differing in antibiotic resistance were selected and subjected to iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics analysis. Differential proteins among the three A. baumannii isolates were further identified and subjected to gene ontology functional enrichment analysis. A resistant isolate (A1), a less resistant one (A8) and a susceptible one (A9) were selected. In total, there were 424 differentially expressed proteins (DEPs) between the A1 and A8 isolates, 1,992 DEPs between the A9 and A1 isolates, and 1,956 DEPs between the A8 and A9 isolates. The upregulation of I6TUC8 and Q0GA83 in the A1 and A8 isolates may be responsible for their higher resistance to ceftriaxone. The higher gentamicin resistance of A. baumannii isolates A1 and A8 when compared to A9 may be related to the higher expression levels of O05286 and D0CCK1, while the higher Q2FCY1 expression level may contribute more to strong gentamicin resistance in A1. The higher levels of L9LWL7, L9MDB0, K9C9W3, E2IGU7, B6E129, G8HYR7, D2XTB0 and D2XTB0 may be responsible for the higher carbapenem resistance of isolate A1 as compared to A8. [ABSTRACT FROM AUTHOR]

The Golgi is the central organelle in the secretory pathway, essential for post-translational modifications, sorting and trafficking of secretory and membrane proteins and lipids in all eukaryotic cells. During mitosis, the mammalian Golgi membranes undergo continuous disassembly and reassembly processes which are critical for Golgi biogenesis during the cell division. To better understand the underlying molecular mechanism of this highly dynamic process, we analyzed the proteins that are in or associated with interphase and mitotic Golgi membranes using an in vitro Golgi assembly assay and quantitative proteomics. In this study, by combining an isobaric mass tag labeling strategy with OFFGEL peptide fractionation, LC-MS/MS analyses identified and quantified a total of 1193 Golgi-resident or -associated proteins. These proteins included Golgi structural proteins, Golgi-resident enzymes, Rab GTPases, and SNARE proteins. This systematic quantitative proteomic study revealed the comprehensive molecular machinery of the Golgi and the dynamic protein changes in its disassembly and reassembly processes. Here we describe the detailed procedures and protocols for this analysis., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine abundances of proteins and peptides in complex biological samples. Several methods have been developed and adapted for accurate quantification based on chemical isotopic labeling. Among various chemical isotopic labeling techniques, isobaric tagging approaches rely on the analysis of peptides from MS2-based quantification rather than MS1-based quantification. In this review, we will provide an overview of several isobaric tags along with some recent developments including complementary ion tags, improvements in sensitive quantitation of analytes with lower abundance, strategies to increase multiplexing capabilities, and targeted analysis strategies. We will also discuss limitations of isobaric tags and approaches to alleviate these restrictions through bioinformatic tools and data acquisition methods. This review will highlight several applications of isobaric tags, including biomarker discovery and validation, thermal proteome profiling, cross-linking for structural investigations, single-cell analysis, top-down proteomics, along with applications to different molecules including neuropeptides, glycans, metabolites, and lipids, while providing considerations and evaluations to each application., (© 2022 The Authors. Proteomics published by Wiley-VCH GmbH.)

STIM1 is an ER/SR transmembrane protein that interacts with ORAI1 to activate store operated Ca 2+ entry (SOCE) upon ER/SR depletion of calcium. Normally highly expressed in skeletal muscle, STIM1 deficiency causes significant changes to mitochondrial ultrastructure that do not occur with loss of ORAI1 or other components of SOCE. The datasets in this article are from large-scale proteomics and phosphoproteomics experiments in an inducible mouse model of skeletal muscle-specific STIM1 knock out (KO). These data reveal statistically significant changes in the relative abundance of specific proteins and sites of protein phosphorylation in STIM1 KO gastrocnemius. Protein samples from five biological replicates of each condition (+/- STIM1) were enzymatically digested, the resulting peptides labeled with tandem mass tag (TMT) reagents, mixed, and fractionated. Phosphopeptides were enriched and a small amount of each input retained for protein abundance analysis. All phosphopeptide and input fractions were analyzed by nano LC-MS/MS on a Q Exactive Plus Orbitrap mass spectrometer, searched with Proteome Discoverer software, and processed with in-house R-scripts for data normalization and statistical analysis. Article published in Molecular Metabolism [1]., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s). Published by Elsevier Inc.)

The transport and translation of dendritic mRNAs by RNA-binding proteins (RBPs) allows for spatially restricted gene expression in neuronal processes. Although local translation in neuronal dendrites is now well documented, there is little evidence for corresponding effects on local synaptic function. Here, we report that the RBP Sam68 promotes the localization and translation of Arc mRNA preferentially in distal dendrites of rodent hippocampal CA1 pyramidal neurons. Consistent with Arc function in translation-dependent synaptic plasticity, we find that Sam68 knockout (KO) mice display impaired metabotropic glutamate-receptor-dependent long-term depression (mGluR-LTD) and impaired structural plasticity exclusively at distal Schaffer-collateral synapses. Moreover, by using quantitative proteomics, we find that the Sam68 interactome contains numerous regulators of mRNA translation and synaptic function. This work identifies an important player in Arc expression, provides a general framework for Sam68 regulation of protein synthesis, and uncovers a mechanism that enables the precise spatiotemporal expression of long-term plasticity throughout neurons. • Sam68 promotes the localization and translation of Arc mRNA at distal dendrites • Distinct processes regulate Arc protein synthesis at the soma versus the dendrite • Sam68 regulates structural and functional plasticity exclusively at distal synapses • Interactome analysis reveals a role for Sam68 in translation initiation Although local translation in neuronal dendrites is well documented, there is little evidence for corresponding effects on local synaptic function. Klein et al. demonstrate that Sam68 is required for Arc protein synthesis at distal dendritic regions and is required for synaptic plasticity exclusively at distal dendrites of hippocampal pyramidal neurons. [ABSTRACT FROM AUTHOR]

Numerous candidate genes related to apomixis have been identified through transcriptomics; however, the molecular mechanism underlying apomixis remains unclear. Elucidation of the underlying mechanisms is essential to expand its application in crop breeding. Therefore, here, we employed the isobaric tags for a relative and absolute quantification labeling technology to investigate the protein expression in Boehmeria tricuspis generated through different reproductive modes at the functional megaspore stage. We identified 40 differential abundance proteins associated with apomeiosis, most of which were involved in "response to stress". Functional analysis suggested that lower levels of reactive oxygen species (ROS) play a role in inducing the development of apomeiosis. Proteins related to ROS regulation, cell wall modifications, and stability under heat stress play a crucial role in the development of diplosporic apomeiosis. Our results give evidence to the insight that stress can induce a switch from apomixis to sexuality by ROS content, and an increased composition of stress tolerance as well as secondary metabolites can buffer ROS effects. Precise coordination of these proteins involved in inter-related regulatory control mechanisms may act together in the transition from the sexual to apomixis development.

Myofibroblasts form adhesions to their underlying extracellular matrices, which is an essential step in their formation and differentiation. These adhesions comprise protein-rich aggregates of a wide variety of signaling, cytoskeletal, cell adhesion, and matrix proteins that interact with one another to enable bidirectional flow of information between the cell and the surrounding extracellular matrix. The concentrated repertoire of the proteins in matrix adhesions of myofibroblasts (i.e., over 450 different proteins) and their important role in regulating the metabolic activities of myofibroblasts, has motivated in-depth analysis of their protein complement and how this repertoire is influenced by experimental conditions.In this protocol I describe in detail: (1) the method for isolating focal adhesion-associated proteins using matrix ligand-bound magnetite beads; (2) the method for eluting the proteins from the beads and their preparation for mass spectrometry (Fig. 1). I also briefly consider the mass spectrometry methods including the use of isobaric tags to enable multifactorial experiments and the analysis of the identified proteins. I consider the advantages of these approaches, and the challenges and pitfalls that are encountered with these methods.

So far, over 50 spontaneous male sterile mutants of tomato have been described and most of them are categorized as genetic male sterility. To date, the mechanism of tomato genetic male sterility remained unclear. In this study, differential proteomic analysis is performed between genetic male sterile line (2-517), which carries the male sterility (ms10 35 ) gene, and its wild-type (VF-11) using isobaric tags for relative and absolute quantification-based strategy. A total of 8272 proteins are quantified in the 2-517 and VF-11 lines at the floral bud and florescence stages. These proteins are involved in different cellular and metabolic processes, which express obvious functional tendencies toward the hydroxylation of the ω-carbon in fatty acids, the tricarboxylic acid cycle, the glycolytic, and pentose phosphate pathways. Based on the results, a protein network explaining the mechanisms of tomato genetic male sterility is proposed, finding the compromising fat acid metabolism may cause the male sterility. These results are confirmed by parallel reaction monitoring, quantitative Real-time PCR (qRT-PCR), and physiological assays. Taken together, these results provide new insights into the metabolic pathway of anther abortion induced by ms10 35 and offer useful clues to identify the crucial proteins involved in genetic male sterility in tomato., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cells respond to environmental perturbations and insults through modulating protein abundance and function. However, the majority of studies have focused on changes in RNA abundance because quantitative transcriptomics has historically been more facile than quantitative proteomics. Modern Orbitrap mass spectrometers now provide sensitive and deep proteome coverage, allowing direct, global quantification of not only protein abundance but also post-translational modifications (PTMs) that regulate protein activity. We implemented and validated using the well-characterized heat shock response of budding yeast, a tandem mass tagging (TMT), triple-stage mass spectrometry (MS 3 ) strategy to measure global changes in the proteome during the yeast heat shock response over nine time points. We report that basic-pH, ultra-high performance liquid chromatography (UPLC) fractionation of tryptic peptides yields superfractions of minimal redundancy, a crucial requirement for deep coverage and quantification by subsequent LC-MS 3 . We quantified 2275 proteins across three biological replicates and found that differential expression peaked near 90 min following heat shock (with 868 differentially expressed proteins at 5% false discovery rate). The sensitivity of the approach also allowed us to detect changes in the relative abundance of ubiquitination and phosphorylation PTMs over time. Remarkably, relative quantification of post-translationally modified peptides revealed striking evidence of regulation of the heat shock response by protein PTMs. These data demonstrate that the high precision of TMT-MS 3 enables peptide-level quantification of samples, which can reveal important regulation of protein abundance and regulatory PTMs under various experimental conditions.

The data-dependent acquisition in mass spectrometry-based proteomics combined with quantitative analysis using isobaric labeling (iTRAQ and TMT) inevitably introduces missing values in proteomic experiments where a number of LC-runs are combined, especially in the growing field of shotgun clinical proteomics, where the protein profiles from the proteomics analysis of several hundred patient samples are compared and correlated to clinical traits such as a specific disease or disease treatment in order to link specific outcomes to one or more proteins. In the context of clinical research it is evident that missing values in such datasets reduce the power of the downstream statistical analysis therefore may hampers the linking of the expression of disease traits to the expression of specific proteins that may be useful for prognostic, diagnostic, or predictive purposes. In our study, we tested three data imputation approaches initially developed for microarray data for the imputation of missing values in datasets that are generated by several runs of shotgun proteomic experiments and where the data were relative protein abundances based on isobaric tags (iTRAQ and TMT). Our conclusion is that imputation methods based on k Nearest Neighbors successfully impute missing values in datasets with up to 50% missing values.

The protein content of amnion is thought to be the primary contributor to its efficacy as a biological dressing for wounds. Protein elution into antibiotic processing media has been reported, but the effect of antiseptic-based processing methods is unknown. Amniotic membranes were collected from eight healthy mares. Samples were collected after removal of gross debris. Tissues were subsequently divided and processed with either 0.05% chlorhexidine or 2% iodine/0.25% acetic acid. After protein extraction and trypsin digestion, the proteins were labeled with 8-plex iTRAQ tags, combined, and analyzed by high-resolution liquid chromatography-mass spectrometry. The MaxQuant-Perseus software suite was used to identify and quantify sample proteins, with functional annotation performed in PANTHER. There were 220 unique proteins identified, of which 144 were found in all individuals and across all conditions, several with a known role in wound healing. Contrary to expectations, processing did not significantly alter the protein content of the amnion tissue. Limitations include the small sample size and single time point. These results suggest that either processing method is acceptable for use in the preparation of equine amnion dressings. The role of expressed proteins in the biological activity of amnion dressings remains to be elucidated.

Despite many years of studies, ovarian cancer remains one of the top ten cancers worldwide. Its high mortality rate is mainly due to lack of sufficient diagnostic methods. For this reason, our research focused on the identification of blood markers whose appearance would precede the clinical manifestation of the disease. ITRAQ-tagging (isobaric Tags for Relative and Absolute Quantification) coupled with mass spectrometry technology was applied. Three groups of samples derived from patients with: ovarian cancer, benign ovarian tumor, and healthy controls, were examined. Mass spectrometry analysis allowed for highlighting the dysregulation of several proteins associated with ovarian cancer. Further validation of the obtained results indicated that five proteins (Serotransferrin, Amyloid A1, Hemopexin, C-reactive protein, Albumin) were differentially expressed in ovarian cancer group. Interestingly, the addition of Albumin, Serotransferrin, and Amyloid A1 to CA125 (cancer antigen 125) and HE4 (human epididymis protein4) improved the diagnostic performance of the model discriminating between benign and malignant tumors. Identified proteins shed light on the molecular signaling pathways that are associated with ovarian cancer development and should be further investigated in future studies. Our findings indicate five proteins with a strong potential to use in a multimarker test for screening and detection of ovarian cancer. [ABSTRACT FROM AUTHOR]

PfPK7 is an "orphan" kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and pfpk7 - parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the pfpk7 - cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates of PfPK7. We demonstrated that PfPK7 is active toward three of these potential novel substrates; however, PfPK7 did not phosphorylate many of the other proteins, suggesting that decreased phosphorylation in these proteins is an indirect effect. Our phosphoproteomics analysis is the first study to identify direct substrates of PfPK7 and reveals potential downstream or compensatory signaling pathways.

Isobaric labeling quantification of peptides has become a method of choice for mass spectrometry-based proteomics studies. However, despite of wide variety of commercially available isobaric tags, none of the currently available methods offers significant improvement of sensitivity of detection during MS experiment. Recently, many strategies were applied to increase the ionization efficiency of peptides involving chemical modifications introducing quaternary ammonium fixed charge. Here, we present a novel quaternary ammonium-based isobaric tag for relative and absolute quantification of peptides (QAS-iTRAQ 2-plex). Upon collisional activation, the new stable benzylic-type cationic reporter ion is liberated from the tag. Deuterium atoms were used to offset the differential masses of a reporter group. We tested the applicability of QAS-iTRAQ 2-plex reagent on a series of model peptides as well as bovine serum albumin tryptic digest. Obtained results suggest usefulness of this isobaric ionization tag for relative and absolute quantification of peptides., (Copyright © 2017 John Wiley & Sons, Ltd.)

Peptidomics is the detection and identification of the peptides present in a sample, while quantitative peptidomics provides additional information about the amounts of these peptides. Comparison of peptide levels among two or more samples is termed relative quantitation. It is also possible to perform absolute quantitation of peptide levels in which the biological sample is compared to synthetic standards, which requires a separate standard for each peptide. In contrast, relative quantitation can compare levels of all peptides that are detectable in a sample, which can exceed 1000 peptides in a complex sample. In this chapter, various techniques used for quantitative peptidomics are described along with discussion of the advantages and disadvantages of each approach. A guide to selecting the optimal quantitative approach is provided, based on the goals of the experiment and the resources that are available.

Isobaric tagging reagents have become an invaluable tool for multiplexed quantitative proteomic analysis. These reagents can label multiple, distinct peptide samples from virtually any source material (e.g., tissue, cell line, purified proteins), allowing users the opportunity to assess changes in peptide abundances across many different time points or experimental conditions. Here, we describe the application of isobaric peptide labeling, specifically 8plex isobaric tags for relative and absolute quantitation (8plex iTRAQ), for quantitative phosphoproteomic analysis of cultured cells or tissue suspensions. For this particular protocol, labeled samples are pooled, fractionated by strong cation exchange chromatography, enriched for phosphopeptides, and analyzed by tandem mass spectrometry (LC-MS/MS) for both peptide identification and quantitation.

Isobaric tags for relative and absolute quantitation (iTRAQ) is a technology that utilizes isobaric reagents to label the primary amines of peptides and proteins and is used in proteomics to study quantitative changes in the proteome by tandem mass spectrometry . Here, we present an adaptation of the iTRAQ experimental protocol for plants that allows the identification and quantitation of more than 12,000 plant proteins in Arabidopsis with a false discovery rate of less than 5 %.

We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.

Mass spectrometry plays a key role in relative quantitative comparisons of proteins in order to understand their functional role in biological systems upon perturbation. In this review, we review studies that examine different aspects of isobaric labeling-based relative quantification for shotgun proteomic analysis. In particular, we focus on different types of isobaric reagents and their reaction chemistry (e.g., amine-, carbonyl-, and sulfhydryl-reactive). Various factors, such as ratio compression, reporter ion dynamic range, and others, cause an underestimation of changes in relative abundance of proteins across samples, undermining the ability of the isobaric labeling approach to be truly quantitative. These factors that affect quantification and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements will be discussed. Finally, the extended application of isobaric labeling-based approach in hyperplexing strategy, targeted quantification, and phosphopeptide analysis are also examined.

Despite the prevalence of diabetes and the global health risks it poses, the biochemical pathogenesis of diabetic complications remains poorly understood with few effective therapies. This study employs capillary liquid chromatography (capLC) and tandem mass spectrometry (MS/MS) in conjunction with both global metabolomics and isobaric tags specific to amines and carbonyls to probe aortic metabolic content in diabetic mice with hyperglycemia, hyperlipidemia, hypertension, and stenotic vascular damage. Using these combined techniques, metabolites well-characterized in diabetes as well as novel pathways were investigated. A total of 53,986 features were detected, 719 compounds were identified as having significant fold changes (thresholds ≥ 2 or ≤ 0.5), and 48 metabolic pathways were found to be altered with at least 2 metabolite hits in diabetic samples. Pathways related to carbonyl stress, carbohydrate metabolism, and amino acid metabolism showed the greatest number of metabolite changes. Three novel pathways with previously limited or undescribed roles in diabetic complications--vitamin B6, propanoate, and butanoate metabolism--were also shown to be altered in multiple points along the pathway. These discoveries support the theory that diabetic vascular complications arise from the interplay of a myriad of metabolic pathways in conjunction with oxidative and carbonyl stress, which may provide not only new and much needed biomarkers but also insights into novel therapeutic targets.

Quantitative proteomics technology based on isobaric tags is playing an important role in proteomic investigation. In this paper, we present an automated software, named IQuant, which integrates a postprocessing tool of protein identification and advanced statistical algorithms to process the MS/MS signals generated from the peptides labeled by isobaric tags and aims at proteomics quantification. The software of IQuant, which is freely downloaded at http://sourceforge.net/projects/iquant/, can run from a graphical user interface and a command-line interface, and can work on both Windows and Linux systems., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)