Mass Spectrometry-based Proteomic Studies Research Articles

Recent Contributions of Proteomics to Our Understanding of Reversible Nε-Lysine Acylation in Bacteria.

Post-translational modifications (PTMs) have been extensively studied in both eukaryotes and prokaryotes. Lysine acetylation, originally thought to be a rare occurrence in bacteria, is now recognized as a prevalent and important PTM in more than 50 species. This expansion in interest in bacterial PTMs became possible with the advancement of mass spectrometry technology and improved reagents such as acyl-modification specific antibodies. In this Review, we discuss how mass spectrometry-based proteomic studies of lysine acetylation and other acyl modifications have contributed to our understanding of bacterial physiology, focusing on recently published studies from 2018 to 2023. We begin with a discussion of approaches used to study bacterial PTMs. Next, we discuss newly characterized acylomes, including acetylomes, succinylomes, and malonylomes, in different bacterial species. In addition, we examine proteomic contributions to our understanding of bacterial virulence and biofilm formation. Finally, we discuss the contributions of mass spectrometry to our understanding of the mechanisms of acetylation, both enzymatic and nonenzymatic. We end with a discussion of the current state of the field and possible future research avenues to explore.

RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis

Alternative splicing is a prevalent gene-regulatory mechanism, with over 95% of multi-exon human genes estimated to be alternatively spliced. Here, we describe a tissue-specific, developmentally regulated, highly conserved, and disease-associated alternative splicing event in exon 7 of the eyes absent homolog 3 (Eya3) gene. We discovered that EYA3 expression is vital to the proliferation and differentiation of myoblasts. Genome-wide transcriptomic analysis and mass spectrometry-based proteomic studies identified SIX homeobox 4 (SIX4) and zinc finger and BTB-domain containing 1 (ZBTB1), as major transcription factors that interact with EYA3 to dictate gene expression. EYA3 isoforms differentially regulate transcription, indicating that splicing aids in temporal control of gene expression during muscle cell differentiation. Finally, we identified RNA-binding fox-1 homolog 2 (RBFOX2) as the main regulator of EYA3 splicing. Together, our findings illustrate the interplay between alternative splicing and transcription during myogenesis.

Mass Spectrometry-Based Proteomics for Discovering Salivary Biomarkers in Periodontitis: A Systematic Review.

Periodontitis is one of the primary causes of tooth loss, and is also related to various systemic diseases. Early detection of this condition is crucial when it comes to preventing further oral damage and the associated health complications. This study offers a systematic review of the literature published up to April 2023, and aims to clearly explain the role of proteomics in identifying salivary biomarkers for periodontitis. Comprehensive searches were conducted on PubMed and Web of Science to shortlist pertinent studies. The inclusion criterion was those that reported on mass spectrometry-driven proteomic analyses of saliva samples from periodontitis cohorts, while those on gingivitis or other oral diseases were excluded. An assessment for risk of bias was carried out using the Newcastle-Ottawa Scale and Quality Assessment of Diagnostic Accuracy Studies or the NIH quality assessment tool, and a meta-analysis was performed for replicable candidate biomarkers, i.e., consistently reported candidate biomarkers (in specific saliva samples, and periodontitis subgroups, reported in ≥2 independent cohorts/reports) were identified. A Gene Ontology enrichment analysis was conducted using the Database for Annotation, Visualization, and Integrated Discovery bioinformatics resources, which consistently expressed candidate biomarkers, to explore the predominant pathway wherein salivary biomarkers consistently manifested. Of the 15 studies included, 13 were case-control studies targeting diagnostic biomarkers for periodontitis participants (periodontally healthy/diseased, n = 342/432), while two focused on biomarkers responsive to periodontal treatment (n = 26 participants). The case-control studies were considered to have a low risk of bias, while the periodontitis treatment studies were deemed fair. Summary estimate and confidence/credible interval, etc. determination for the identified putative salivary biomarkers could not be ascertained due to the low number of studies in each case. The results from the included case-control studies identified nine consistently expressed candidate biomarkers (from nine studies with 230/297 periodontally healthy/diseased participants): (i) those that were upregulated: alpha-amylase, serum albumin, complement C3, neutrophil defensin, profilin-1, and S100-P; and (ii) those that were downregulated: carbonic anhydrase 6, immunoglobulin J chain, and lactoferrin. All putative biomarkers exhibited consistent regulation patterns. The implications of the current putative marker proteins identified were reviewed, with a focus on their potential roles in periodontitis diagnosis and pathogenesis, and as putative therapeutic targets. Although in its early stages, mass spectrometry-based salivary periodontal disease biomarker proteomics detection appeared promising. More mass spectrometry-based proteomics studies, with or without the aid of already available clinical biochemical approaches, are warranted to aid the discovery, identification, and validation of periodontal health/disease indicator molecule(s). Protocol registration number: CRD42023447722; supported by RD-02-202410 and GRF17119917.

Identification of novel serum protein biomarkers in the context of 3P medicine for intravenous leiomyomatosis: a data-independent acquisition mass spectrometry-based proteomics study

Identification of novel serum protein biomarkers in the context of 3P medicine for intravenous leiomyomatosis: a data-independent acquisition mass spectrometry-based proteomics study

Mechanisms of cardiac myosin replacement.

A recent mass spectrometry-based proteomic study from our lab demonstrated that newly synthesized myosin molecules are randomly inserted into preexisting thick filaments in adult mouse hearts in vivo. Therefore, we hypothesized that myosin molecules exist in a dynamic equilibrium between a filamentous pool required for contractility, and a monomeric pool required for myosin replacement. To test this hypothesis, we replaced 27 ± 7% of the cardiac myosin regulatory light chain (RLC) in adult mice with fluorescent RLC (GFP-RLC), via adeno-associated viral transfection. We visualized the random distribution of GFP-RLC within thick filaments in intact hearts with 2-photon microscopy, and the exchange of GFP-RLC between these filaments by quantifying fluorescence recovery after photobleaching (FRAP). Rapid FRAP occurred within minutes, when photobleaching small, ∼1-µm3 areas, of partial sarcomeres. These data demonstrate that the GFP-RLC exchanges between the thick filaments and a monomeric pool on a timescale that is orders of magnitude faster than that of protein replacement (t1/2 = 10.8 days). To determine whether the mechanism of this exchange may involve the folding of monomeric myosin, GFP-labeled myosin molecules were extracted from the transfected hearts and their Stokes radii were measured over a range of ionic strengths, using microfluidic diffusional sizing. At low ionic strengths (150 mM KCl), filament formation was favored, but the monomeric fraction of GFP-labeled myosin demonstrated a radius of 12.6±1.1 nm. At higher ionic strengths (400-600 mM KCl), the radius was increased to 18.0 nm. Similar radii have been reported for smooth muscle myosin monomers in folded and extended conformations. Together, these data suggest that myosin molecules exist in rapid equilibrium between a filamentous and monomeric pool within the sarcomere, and individual monomers may adopt a folded, compact conformation to regulate their exchange into, and out of filaments.

Proteomic Characterisation of the Plasma Proteome in Extramedullary Multiple Myeloma Identifies Potential Prognostic Biomarkers

Introduction: Extramedullary multiple myeloma (EMM) is an aggressive manifestation of multiple myeloma (MM) reported to occur in approximately 7% of patients at diagnosis and up to 30% of patients at relapse. EMM is characterised by the spread of malignant plasma cells from the bone marrow microenvironment to distal tissues or organs. It is associated with an adverse prognosis, correlating with a significant reduction in overall survival. Currently there are no validated, established biomarkers to predict EMM. Furthermore, EMM is often treated similarly to high-risk MM with no targeted therapeutic strategies. In-depth proteomic studies on EMM are lacking and the underlying molecular mechanisms that facilitate extramedullary transition are yet to be fully defined. Novel biomarkers and therapeutic targets are urgently required. To enhance our understanding of EMM and to identify novel prognostic biomarkers, we performed a mass spectrometry-based proteomic study on plasma from MM patients with and without extramedullary spread. Methods: Label-free liquid chromatography mass spectrometric analysis of age and gender-matched medullary MM (n=8) and EMM (n=9) blood plasma samples was carried out using a Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific). Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identification of peptides and proteins. Several parameters were defined for protein identification: MS/MS mass tolerance was set to 0.02 Da; peptide mass tolerance was set to 10ppm; methionine oxidation was set as a variable modification; carbamido-methylation was set as a fixed modification; and up to two missed cleavages were tolerated. Peptide probability was set to high confidence. Data was imported into Perseus (1.6.14.0) for further analysis. Proteins with less than 70% valid values were removed from the analysis. Proteins of interest were identified based on an FDR-adjusted p-value ≤0.1, fold change >1.5 between experimental groups. Six proteins were selected for further validation using DuoSet enzyme linked immunosorbent assay (ELISA) kits (R&D Systems). We performed receiver operating characteristic (ROC) and area under the curve (AUC) analyses to determine the diagnostic potential of the validated proteins. Results: The median age was 65. Survival analysis revealed a statistically significant change in overall survival (OS) between the two patient cohorts (Log-rank = 3.977, P = 0.046). The median OS of patients with EMM and those without extramedullary spread was 19 months and 83 months, respectively. Our quantitative MS-based proteomic analysis identified 21 proteins of differential abundance between EMM and MM patient plasma (False discovery rate (FDR)-adjusted p-value < 0.1, fold change > 1.5) (Fig. 1A). Antibody-based validation using ELISAs was performed on six proteins (vascular cell adhesion molecule 1 (VCAM1), hepatocyte growth factor activator (HGFA), pigment epithelial-derived factor (PEDF), alpha-2-macroglobulin (A2M), cholinesterase (BCHE), aminopeptidase N (CD13)). VCAM1, HGFA and PEDF were confirmed as being significantly altered between the two cohorts (FDR-adjusted p-value < 0.05). VCAM1, HGFA and PEDF were subject to ROC analyses, demonstrating high discriminatory power for EMM diagnosis (AUC = 0.96, AUC = 0.85, and AUC = 0.97, respectively). The diagnostic efficacy was further enhanced by combining these biomarkers using a logistic regression model (AUC = 1). Conclusion: Our mass spectrometry and antibody-based study identified proteins of differential abundance in the blood plasma of MM patients with and without extramedullary spread. VCAM1, PEDF and HGFA represent promising predictive biomarkers and warrant further investigation in a larger cohort of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proteomic profiling of impaired excitation-contraction coupling and abnormal calcium handling in muscular dystrophy.

The X-linked inherited neuromuscular disorder Duchenne muscular dystrophy is characterised by primary abnormalities in the membrane cytoskeletal component dystrophin. The almost complete absence of the Dp427-M isoform of dystrophin in skeletal muscles renders contractile fibres more susceptible to progressive degeneration and a leaky sarcolemma membrane. This in turn results in abnormal calcium homeostasis, enhanced proteolysis and impaired excitation-contraction coupling. Biochemical and mass spectrometry-based proteomic studies of both patient biopsy specimens and genetic animal models of dystrophinopathy have demonstrated significant changes in the concentration and/or physiological function of essential calcium-regulatory proteins in dystrophin-lacking voluntary muscles. Abnormalities include dystrophinopathy-associated changes in voltage sensing receptors, calcium release channels, calcium pumps and calcium binding proteins. This review article provides an overview of the importance of the sarcolemmal dystrophin-glycoprotein complex and the wider dystrophin complexome in skeletal muscle and its linkage to depolarisation-induced calcium-release mechanisms and the excitation-contraction-relaxation cycle. Besides chronic inflammation, fat substitution and reactive myofibrosis, a major pathobiochemical hallmark of X-linked muscular dystrophy is represented by the chronic influx of calcium ions through the damaged plasmalemma in conjunction with abnormal intracellular calcium fluxes and buffering. Impaired calcium handling proteins should therefore be included in an improved biomarker signature of Duchenne muscular dystrophy.

Exploiting Interdata Relationships in Prostate Cancer Proteomes: Clinical Significance of HO-1, HSPB1, DDB1 and 14-3-3ζ/δ Interaction

PURPOSE Prostate cancer (PCa) is the second most common type of cancer in men and the sixth leading cause of cancer-related death in men worldwide. The discovery of new therapeutic avenues in PCa, and the development of effective drugs in the era of personalized medicine, would greatly benefit from the field of proteomics. We have previously shown the anti-tumoral role of Heme oxygenase 1 (HO-1) in PCa. METHODS In this work, we undertook a mass spectrometry-based proteomics study to assess whether in PCa cells and under oxidative stress conditions, HO-1 could interact with proteins previously documented to have nuclear localization. RESULTS Among the HO-1 interactors, we identified 11 proteins with nuclear localization. Significant and positive correlation between HMOX1 and six of those genes was observed, using the GSE70770 dataset. Alternatively, HMOX1 and YWHAZ showed negative correlation. High expression of HNRNPA2B1, HSPB1, NPM1, DDB1, HMGA1, ZC3HAV1, and HMOX1 was associated with an increased relapse-free survival (RFS) in PCa patients through univariable analyses. Further, PCa patients with high HSPB1/HMOX1, DDB1/HMOX1, and YWHAZ/HMOX1 showed a worse RFS compared with patients with lower ratios. Moreover, by using a prognostic risk score model, a decrease in RFS for patients with higher scores of this signature was observed. However, the only factor significantly associated with a higher risk of relapse was high YWHAZ. HSPB1, DDB1 and YWHAZ independence from PCa clinic-pathological parameters was confirmed through multivariable analyses. Moreover, co-immunoprecipitation analysis in PCa cells ascertained HO-1/14-3-3ζ/δ (YWHAZ encoding gene) interaction. CONCLUSION Herein, we report novel interactions between HO-1 and HSPB1, DDB1 and 14-3-3ζ/δ, highlighting their clinical relevance in PCa.

Exploiting Interdata Relationships in Prostate Cancer Proteomes: Clinical Significance of HO-1 Interactors.

Prostate cancer (PCa) cells display abnormal expression of proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown the anti-tumoral role of heme oxygenase 1 (HO-1) in this disease. In this work, we undertook a mass spectrometry-based proteomics study to identify HO-1 molecular interactors that might collaborate with its modulatory function in PCa. Among the HO-1 interactors, we identified proteins with nuclear localization. Correlation analyses, using the PCa GSE70770 dataset, showed a significant and positive correlation between HMOX1 and 6 of those genes. Alternatively, HMOX1 and YWHAZ showed a negative correlation. Univariable analyses evidenced that high expression of HNRNPA2B1, HSPB1, NPM1, DDB1, HMGA1, ZC3HAV1, and HMOX1 was associated with increased relapse-free survival (RFS) in PCa patients. Further, PCa patients with high HSPB1/HMOX1, DDB1/HMOX1, and YWHAZ/HMOX1 showed a worse RFS compared with patients with lower ratios. Moreover, a decrease in RFS for patients with higher scores of this signature was observed using a prognostic risk score model. However, the only factor significantly associated with a higher risk of relapse was high YWHAZ. Multivariable analyses confirmed HSPB1, DDB1, and YWHAZ independence from PCa clinic-pathological parameters. In parallel, co-immunoprecipitation analysis in PCa cells ascertained HO-1/14-3-3ζ/δ (protein encoded by YWHAZ) interaction. Herein, we describe a novel protein interaction between HO-1 and 14-3-3ζ/δ in PCa and highlight these factors as potential therapeutic targets.

Integrative Multi-omics Landscape of Non-structural Protein 3 of Severe Acute Respiratory Syndrome Coronaviruses

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is currently a global pandemic. Extensive investigations have been performed to study the clinical and cellular effects of SARS-CoV-2 infection. Mass spectrometry-based proteomics studies have revealed the cellular changes due to the infection and identified a plethora of interactors for all SARS-CoV-2 components, except for the longest non-structural protein 3 (NSP3). Here, we expressed the full-length NSP3 proteins of SARS-CoV and SARS-CoV-2 to investigate their unique and shared functions using multi-omics methods. We conducted interactome, phosphoproteome, ubiquitylome, transcriptome, and proteome analyses of NSP3-expressing cells. We found that NSP3 plays essential roles in cellular functions such as RNA metabolism and immune response (e.g., NF-κB signal transduction). Interestingly, we showed that SARS-CoV-2 NSP3 has both endoplasmic reticulum and mitochondrial localizations. In addition, SARS-CoV-2 NSP3 is more closely related to mitochondrial ribosomal proteins, whereas SARS-CoV NSP3 is related to the cytosolic ribosomal proteins. In summary, our integrative multi-omics study of NSP3 improves the understanding of the functions of NSP3 and offers potential targets for the development of anti-SARS strategies.

Optimization of proteomics sample preparation for forensic analysis of skin samples

We present an efficient protein extraction and in-solution enzymatic digestion protocol optimized for mass spectrometry-based proteomics studies of human skin samples. Human skin cells are a proteinaceous matrix that can enable forensic identification of individuals. We performed a systematic optimization of proteomic sample preparation for a protein-based human forensic identification application. Digestion parameters, including incubation duration, temperature, and the type and concentration of surfactant, were systematically varied to maximize digestion completeness. Through replicate digestions, parameter optimization was performed to maximize repeatability and increase the number of identified peptides and proteins. Final digestion conditions were selected based on the parameters that yielded the greatest percent of peptides with zero missed tryptic cleavages, which benefit the analysis of genetically variable peptides (GVPs). We evaluated the final digestion conditions for identification of GVPs by applying MS-based proteomics on a mixed-donor sample. The results were searched against a human proteome database appended with a database of GVPs constructed from known non-synonymous single nucleotide polymorphisms (SNPs) that occur at known population frequencies. The aim of this study was to demonstrate the potential of our proteomics sample preparation for future implementation of GVP analysis by forensic laboratories to facilitate human identification. SignificanceGenetically variable peptides (GVPs) can provide forensic evidence that is complementary to traditional DNA profiling and be potentially used for human identification. An efficient protein extraction and reproducible digestion method of skin proteins is a key contributor for downstream analysis of GVPs and further development of this technology in forensic application. In this study, we optimized the enzymatic digestion conditions, such as incubation time and temperature, for skin samples. Our study is among the first attempts towards optimization of proteomics sample preparation for protein-based skin identification in forensic applications such as touch samples. Our digestion method employs RapiGest (an acid-labile surfactant), trypsin enzymatic digestion, and an incubation time of 16 h at 37 °C.

Cellular Prion Protein Mediates α-Synuclein Uptake, Localization, and Toxicity In Vitro and In Vivo.

The cellular prion protein (PrPC ) is a membrane-bound, multifunctional protein mainly expressed in neuronal tissues. Recent studies indicate that the native trafficking of PrPC can be misused to internalize misfolded amyloid beta and α-synuclein (aSyn) oligomers. We define PrPC 's role in internalizing misfolded aSyn in α-synucleinopathies and identify further involved proteins. We performed comprehensive behavioral studies on four transgenic mouse models (ThySyn and ThySynPrP00, TgM83 and TgMPrP00) at different ages. We developed PrPC -(over)-expressing cell models (cell line and primary cortical neurons), used confocal laser microscopy to perform colocalization studies, applied mass spectrometry to identify interactomes, and determined disassociation constants using surface plasmon resonance (SPR) spectroscopy. Behavioral deficits (memory, anxiety, locomotion, etc.), reduced lifespans, and higher oligomeric aSyn levels were observed in PrPC -expressing mice (ThySyn and TgM83), but not in homologous Prnp ablated mice (ThySynPrP00 and TgMPrP00). PrPC colocalized with and facilitated aSyn (oligomeric and monomeric) internalization in our cell-based models. Glimepiride treatment of PrPC -overexpressing cells reduced aSyn internalization in a dose-dependent manner. SPR analysis showed that the binding affinity of PrPC to monomeric aSyn was lower than to oligomeric aSyn. Mass spectrometry-based proteomic studies identified clathrin in the immunoprecipitates of PrPC and aSyn. SPR was used to show that clathrin binds to recombinant PrP, but not aSyn. Experimental disruption of clathrin-coated vesicles significantly decreased aSyn internalization. PrPC 's native trafficking can be misused to internalize misfolded aSyn through a clathrin-based mechanism, which may facilitate the spreading of pathological aSyn. Disruption of aSyn-PrPC binding is, therefore, an appealing therapeutic target in α-synucleinopathies. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Proteomics Mapping of the ISGylation Landscape in Innate Immunity.

During infection, pathogen sensing and cytokine signaling by the host induce expression of antimicrobial proteins and specialized post-translational modifications. One such protein is ISG15, a ubiquitin-like protein (UBL) conserved among vertebrates. Similar to ubiquitin, ISG15 covalently conjugates to lysine residues in substrate proteins in a process called ISGylation. Mice deficient for ISGylation or lacking ISG15 are strongly susceptible to many viral pathogens and several intracellular bacterial pathogens. Although ISG15 was the first UBL discovered after ubiquitin, the mechanisms behind its protective activity are poorly understood. Largely, this stems from a lack of knowledge on the ISG15 substrate repertoire. To unravel the antiviral activity of ISG15, early studies used mass spectrometry-based proteomics in combination with ISG15 pulldown. Despite reporting hundreds of ISG15 substrates, these studies were unable to identify the exact sites of modification, impeding a clear understanding of the molecular consequences of protein ISGylation. More recently, a peptide-based enrichment approach revolutionized the study of ubiquitin allowing untargeted discovery of ubiquitin substrates, including knowledge of their exact modification sites. Shared molecular determinants between ISG15 and ubiquitin allowed to take advantage of this technology for proteome-wide mapping of ISG15 substrates and modification sites. In this review, we provide a comprehensive overview of mass spectrometry-based proteomics studies on protein ISGylation. We critically discuss the relevant literature, compare reported substrates and sites and make suggestions for future research.

Isoform-specific dysregulation of AMP-activated protein kinase signaling in a non-human primate model of Alzheimer's disease

AMP-activated protein kinase (AMPK) is a molecular sensor that is critical for the maintenance of cellular energy homeostasis, disruption of which has been indicated in multiple neurodegenerative diseases including Alzheimer's disease (AD). Mammalian AMPK is a heterotrimeric complex and its enzymatic α subunit exists in two isoforms: AMPKα1 and AMPKα2. Here we took advantage of a recently characterized non-human primate (NHP) model with sporadic AD-like neuropathology to explore potential relationships between AMPK signaling and AD-like neuropathology. Subjects were nine female vervet monkeys aged 19.5 to 23.4 years old. Subjects were classified into three groups, control lacking AD pathology (n = 3), moderate AD pathology (n = 3), and more severe AD Pathology (n = 3). We found increased activity (assessed by phosphorylation) of AMPKα2 in hippocampi of NHP with AD-like neuropathology, compared to the subjects without AD pathology, with no alterations of AMPKα1 activity. Across all subjects, CSF Abeta42 was inversely associated with cerebral amyloid plaque density. Further, Aβ plaque burden is correlated with levels of either soluble or insoluble brain Aβ measurement. Unbiased mass spectrometry based proteomics studies combined with bioinformatics analysis revealed that many of the dysregulated proteins characteristic of AD neuropathology are associated with AMPK signaling. Our findings on the AMPK molecular signaling cascades provide further support for use of the NHP model to investigate new therapeutic strategies and development of novel biomarkers for Alzheimer's disease.

Statistical Characterization of the Multi-Charged Fragment Ions in the CID and HCD Spectrum

Collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) are the widely used fragmentation technique in mass spectrometry-based proteomics studies. Understanding the fragmentation pattern from the tandem mass spectra using statistical methods helps to implement efficient spectrum analysis algorithms. The study characterizes the frequency of occurrence of multi-charged fragment ions and their neutral loss events of doubly and triply charged peptides in the CID and HCD spectrum. The dependency of the length of the fragment ion on the occurrence of multi-charged fragment ion is characterized here. Study shows that the singly charged fragment ions are generally dominated in the doubly charged peptide spectrum. However, as the length of the product ion increases, the frequency of occurrence of charge 2 fragment ions increases. The y- ions have more tendencies to generate charge 2 fragment ions than b- ions, both in CID and HCD spectrum. The frequency of occurrence of charge 2 fragment ion peaks is prominent upon the dissociation of the triply charged peptides. For triply charged peptides, product ion of higher length occurred in multiple charge states in CID spectrum. The neutral loss peaks mostly exist in charge 2 states in the triply charged peptide spectrum. The b-ions peaks are observed in much less frequency than y-ions in HCD spectrum as the length of the fragment increases. Isotopic peaks are occurred in charge 2 state both in doubly and triply charged peptide’s HCD spectrum.

Proteomic Analyses of Vitreous in Proliferative Diabetic Retinopathy: Prior Studies and Future Outlook.

Vitreous fluid is becoming an increasingly popular medium for the study of retinal disease. Numerous studies have demonstrated that proteomic analysis of the vitreous from patients with proliferative diabetic retinopathy yields valuable molecular information regarding known and novel proteins and pathways involved in this disease. However, there is no standardized methodology for vitreous proteomic studies. Here, we share a suggested protocol for such studies and outline the various experimental and analytic methods that are currently available. We also review prior mass spectrometry-based proteomic studies of the vitreous from patients with proliferative diabetic retinopathy, discuss common pitfalls of these studies, and propose next steps for moving the field forward.

Phytochrome Mediated Responses in Agrobacterium fabrum: Growth, Motility and Plant Infection

The soil bacterium and plant pathogen Agrobacterium fabrum C58 has two phytochrome photoreceptors, Agp1 and Agp2. We found that plant infection and tumor induction by A. fabrum is down-regulated by light and that phytochrome knockout mutants of A. fabrum have diminished infection rates. The regulation pattern of infection matches with that of bacterial conjugation reported earlier, suggesting similar regulatory mechanisms. In the regulation of conjugation and plant infection, phytochromes are active in darkness. This is a major difference to plant phytochromes, which are typically active after irradiation. We also found that propagation and motility were affected in agp1− and agp2− knockout mutants, although propagation was not always affected by light. The regulatory patterns can partially but not completely be explained by modulated histidine kinase activities of Agp1 and Agp2. In a mass spectrometry-based proteomic study, 24 proteins were different between light and dark grown A. fabrum, whereas 382 proteins differed between wild type and phytochrome knockout mutants, pointing again to light independent roles of Agp1 and Agp2.

A Comparative Analysis of Erythropoietin and Carbamoylated Erythropoietin Proteome Profiles.

In recent years, erythropoietin (EPO) has emerged as a useful neuroprotective and neurotrophic molecule that produces antidepressant and cognitive-enhancing effects in psychiatric disorders. However, EPO robustly induces erythropoiesis and elevates red blood cell counts. Chronic administration is therefore likely to increase blood viscosity and produce adverse effects in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered modification of EPO, is non-erythropoietic but retains the neurotrophic and neurotrophic activity of EPO. Blood profile analysis after EPO and CEPO administration showed that CEPO has no effect on red blood cell or platelet counts. We conducted an unbiased, quantitative, mass spectrometry-based proteomics study to comparatively investigate EPO and CEPO-induced protein profiles in neuronal phenotype PC12 cells. Bioinformatics enrichment analysis of the protein expression profiles revealed the upregulation of protein functions related to memory formation such as synaptic plasticity, long term potentiation (LTP), neurotransmitter transport, synaptic vesicle priming, and dendritic spine development. The regulated proteins, with roles in LTP and synaptic plasticity, include calcium/calmodulin-dependent protein kinase type 1 (Camk1), Synaptosomal-Associated Protein, 25 kDa (SNAP-25), Sectretogranin-1 (Chgb), Cortactin (Cttn), Elongation initiation factor 3a (Eif3a) and 60S acidic ribosomal protein P2 (Rplp2). We examined the expression of a subset of regulated proteins, Cortactin, Grb2 and Pleiotrophin, by immunofluorescence analysis in the rat brain. Grb2 was increased in the dentate gyrus by EPO and CEPO. Cortactin was induced by CEPO in the molecular layer, and pleiotrophin was increased in the vasculature by EPO. The results of our study shed light on potential mechanisms whereby EPO and CEPO produce cognitive-enhancing effects in clinical and preclinical studies.

SWATH-MS proteomics of PANC-1 and MIA PaCa-2 pancreatic cancer cells allows identification of drug targets alternative to MEK and PI3K inhibition

Pancreatic cancer remains one of the most lethal diseases with dismal five-year survival rates. Although mutant KRas protein-driven activation of downstream MAPK Raf/MEK/ERK and PI3K/Akt signaling pathways represent major oncogenic alterations, signaling blockade with MEK and PI3K inhibitors has shown that intrinsic resistance may hamper the effectiveness of this targeted approach. However, there have been no mass spectrometry-based proteomic studies for in-depth comparison of protein expression differences between pancreatic cancer cells with sensitivity and resistance to MEK and PI3K kinase inhibitors. In this work, we compared PANC-1 and MIA PaCa-2 pancreatic cancer cells which are, respectively, resistant and sensitive to MEK- and PI3K-targeted therapy. We conducted a label-free data-independent acquisition mass spectrometry (SWATH-MS) study with extensive peptide fractionation to quantitate 4808 proteins and analyze differential expression of 743 proteins between resistant and sensitive cells. This allowed identification of the tumor suppressor protein phosphatase 2A (PP2A) and proteins from mitochondrial respiratory complex I implicated in oxidative phosphorylation as alternative candidate drug targets for cells resistant to MEK and PI3K inhibition. PP2A activator DT-061 decreased viability of PANC-1 cells and this was accompanied by reduced expression of c-Myc. PANC-1 cells also showed response to metformin and the novel complex I inhibitor IACS-010759. These findings provide insights into the distinct cellular proteomes and point out alternative pharmacological targets for MEK and PI3K inhibition-resistant pancreatic cancer cells.

Global Proteome Profiling to Assess Changes in Protein Abundance Using Isobaric Labeling and Liquid Chromatography-Tandem Mass Spectrometry.

Protein degradation is a critical component of all facets of cell biology, and recently methods have been developed to make use of targeted protein degradation as both an investigative tool and a potential therapeutic avenue. Mass spectrometry-based proteomic studies have allowed detailed characterization of changes in protein level and the biology underlying growth, development, and disease. Current methods and instrumentation allow identification and quantitative analysis of thousands of proteins in a single assay. The method described here involves cell lysis and digestion to peptides, labeling peptides with isobaric tagging TMT reagents, basic reversed phase fractionation, and liquid chromatography-tandem mass spectrometry analysis of the enriched peptides.