Quantitative Proteomics Platform Research Articles

Comparative Seed Proteome Profile Reveals No Alternation of Major Allergens in High-Yielding Mung Bean Cultivars.

Mung bean contains up to 32.6% protein and is one of the great sources of plant-based protein. Because many allergens also function as defense-related proteins, it is important to determine their abundance levels in the high-yielding, disease-resistant cultivars. In this study, for the first time, we compared the seed proteome of high-yielding mung bean cultivars developed by a conventional breeding approach. Using a label-free quantitative proteomic platform, we successfully identified and quantified a total of 1373 proteins. Comparative analysis between the high-yielding disease-resistant cultivar (MC5) and the other three cultivars showed that a total of 69 common proteins were significantly altered in their abundances across all cultivars. Bioinformatic analysis of these altered proteins demonstrated that PDF1 (a defensin-like protein) exhibited high sequence similarity and epitope matching with the established peanut allergens, indicating a potential mung bean allergen that showed a cultivar-specific response. Conversely, known mung bean allergen proteins such as PR-2/PR-10 (Vig r 1), Vig r 2, Vig r 4, LTP1, β-conglycinin, and glycinin G4 showed no alternation in the MC5 compared to other cultivars. Taken together, our findings suggest that the known allergen profiles may not be impacted by the conventional plant breeding method to develop improved mung bean cultivars.

Urine proteome profile of firefighters with exposure to emergency fire-induced smoke: A pilot study to identify potential carcinogenic effects

Firefighters are frequently exposed to a variety of chemicals formed from smoke, which pose a risk for numerous diseases, including cancer. Comparative urine proteome profiling could significantly improve our understanding of the early detection of potential cancer biomarkers. In this study, for the first time, we conducted a comparative protein profile analysis of 20 urine samples collected from ten real-life firefighters prior to and following emergency fire-induced smoke. Using a label-free quantitative proteomics platform, we identified and quantified 1325 unique protein groups, of which 45 proteins showed differential expressions in abundance in response to fire-smoke exposure (post) compared to the control (pre). Pathway analysis showed proteins associated with epithelium development (e.g., RHCG, HEG1, ADAMTSL2) and Alzheimer's disease (SORL1) were significantly increased in response to smoke exposure samples. A protein-protein-network study showed a possible link between these differentially abundant proteins and the known cancer gene (TP53). Moreover, a cross-comparison analysis revealed that seven proteins—ALDH1A1, APCS, POMC, COL2A1, RDX, DDAH2, and SDC4 overlapped with the previously published urine cancer proteome datasets, suggesting a potential cancer risk. Our findings demonstrated that the discovery proteomic platform is a promising analytical technique for identifying potential non-invasive biomarkers associated with fire-smoke exposure in firefighters that may be related to cancer.

Chemoproteomic Profiling Maps Zinc-Dependent Cysteine Reactivity.

As a vital micronutrient, zinc is integral to the structure, function, and signaling networks of diverse proteins. Dysregulated zinc levels, due to either excess intake or deficiency, are associated with a spectrum of health disorders. In this context, understanding zinc-regulated biological processes at the molecular level holds significant relevance to public health and clinical practice. Identifying and characterizing zinc-regulated proteins in their diverse proteoforms, however, remain a difficult task in advancing zinc biology. Herein, we address this challenge by developing a quantitative chemical proteomics platform that globally profiles the reactivities of proteinaceous cysteines upon cellular zinc depletion. Exploiting a protein-conjugated resin for the selective removal of Zn2+ from culture media, we identify an array of zinc-sensitive cysteines on proteins with diverse functions based on their increased reactivity upon zinc depletion. Notably, we find that zinc regulates the enzymatic activities, post-translational modifications, and subcellular distributions of selected target proteins such as peroxiredoxin 6 (PRDX6), platelet-activating factor acetylhydrolase IB subunit alpha1 (PAFAH1B3), and phosphoglycerate kinase (PGK1).

Establishment of Quantitative Proteomics Platform for Urine Biomarker Discovery and Validation

Establishment of Quantitative Proteomics Platform for Urine Biomarker Discovery and Validation

Physiological and Differential Proteomic Analysis at Seedling Stage by Induction of Heavy-Ion Beam Radiation in Wheat Seeds.

Novel genetic variations can be obtained by inducing mutations in the plant which help to achieve novel traits. The useful mutant can be obtained through radiation mutation in a short period which can be used as a new material to produce new varieties with high yield and good quality wheat. In this paper, the proteomic analysis of wheat treated with different doses of 12C and 7Li ion beam radiation at the seedling stage was carried out through a Tandem Mass Tag (TMT) tagging quantitative proteomic analysis platform based on high-resolution liquid chromatography-mass spectrometry, and the traditional 60Co-γ-ray radiation treatment for reference. A total of 4,764 up-regulated and 5,542 down-regulated differentially expressed proteins were identified. These proteins were mainly enriched in the KEGG pathway associated with amino acid metabolism, fatty acid metabolism, carbon metabolism, photosynthesis, signal transduction, protein synthesis, and DNA replication. Functional analysis of the differentially expressed proteins showed that the oxidative defense system in the plant defense system was fully involved in the defense response after 12C ion beam and 7Li ion beam radiation treatments. Photosynthesis and photorespiration were inhibited after 12C ion beam and 60Co-γ-ray irradiation treatments, while there was no effect on the plant with 7Li ion beam treatment. In addition, the synthesis of biomolecules such as proteins, as well as multiple signal transduction pathways also respond to radiations. Some selected differentially expressed proteins were verified by Parallel Reaction Monitoring (PRM) and qPCR, and the experimental results were consistent with the quantitative results of TMT. The present study shows that the physiological effect of 12C ion beam radiation treatment is different as compared to the 7Li ion beam, but its similar to the 60Co-γ ray depicting a significant effect on the plant by using the same dose. The results of this study will provide a theoretical basis for the application of 12C and 7Li ion beam radiation in the mutation breeding of wheat and other major crops and promote the development of heavy ion beam radiation mutation breeding technology.

Global proteome response to Pb(II) toxicity in poplar using SWATH-MS-based quantitative proteomics investigation

Lead (Pb) toxicity is a growing serious environmental pollution that threatens human health and crop productivity. Poplar, as an important economic and ecological forest species, has the characteristics of fasting growth and accumulating heavy metals, which is a powerful model plant for phytoremediation. Here, a novel label-free quantitative proteomic platform of SWATH-MS was applied to detect proteome changes in poplar seedling roots following Pb treatment. In total 4388 unique proteins were identified and quantified, among which 542 proteins showed significant abundance changes upon Pb(II) exposure. Functional categorizations revealed that differentially expressed proteins (DEPs) primarily distributed in specialized biological processes. Particularly, lignin and flavonoid biosynthesis pathway were strongly activated upon Pb exposure, implicating their potential roles for Pb detoxification in poplar. Furthermore, hemicellulose and pectin related cell wall proteins exhibited increased abundances, where may function as a sequestration reservoir to reduce Pb toxicity in cytoplasm. Simultaneously, up-regulation of glutathione metabolism may serve as a protective role for Pb-induced oxidative damages in poplar. Further correlation investigation revealed an extra layer of post-transcriptional regulation during Pb response in poplar. Overall, our work represents multiply potential regulators in mediating Pb tolerance in poplar, providing molecular targets and strategies for phytoremediation.

Proteomics Landscape of Alzheimer's Disease.

Alzheimer’s disease (AD) is the most prevalent form of dementia, and the numbers of AD patients are expected to increase as human life expectancy improves. Deposition of β-amyloid protein (Aβ) in the extracellular matrix and intracellular neurofibrillary tangles are molecular hallmarks of the disease. Since the precise pathophysiology of AD has not been elucidated yet, effective treatment is not available. Thus, understanding the disease pathology, as well as identification and development of valid biomarkers, is imperative for early diagnosis as well as for monitoring disease progression and therapeutic responses. Keeping this goal in mind several studies using quantitative proteomics platform have been carried out on both clinical specimens including the brain, cerebrospinal fluid (CSF), plasma and on animal models of AD. In this review, we summarize the mass spectrometry (MS)-based proteomics studies on AD and discuss the discovery as well as validation stages in brief to identify candidate biomarkers.

Loss of H3K27 Methylation Identifies Poor Outcome in Adult-Onset Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an epigenetically heterogeneous disease. The intensity of treatment is currently guided by cytogenetic and molecular genetic risk classifications; however these incompletely predict outcomes, requiring additional information for more accurate predictions. We aimed to identify potential prognostic implications of epigenetic modification of histone proteins, with a focus of H3K27 methylation in relation to mutations in chromatin, splicing and transcriptional regulators. Material and methods: Histone methylation mark expressions were evaluated in a cohort of 241 AML bone marrow (BM) and peripheral blood (PB) samples from patients admitted at the MD Anderson Cancer Center relative to their expression in CD34+ BM derived samples from healthy donors. Simultaneous analysis of 230 proteins was performed using the reverse phase protein array - a high-throughput, quantitative proteomic platform that enables identification of aberrant expressed proteins and the pathways they act in. Additional mutational analysis was performed on 65 BM samples. Results:H3K27Me3 was significantly lower in both BM and PB leukemic-derived samples compared to their expression in normal BM (figure 1A). A greater loss of H3K27Me3 associated with increased proliferative potential and shorter overall survival (OS) in the whole patient population (n=241, HR=0.64, 95% CI=0.47-0.87, p<0.01), as well as in subsets, e.g. cytogenetically normal AML (n=110, HR=0.62, 95% CI=0.40-0.97, p=0.03). To study the prognostic impact of H3K27Me3 in the context of cytogenetic aberrations and mutations, multivariate cox regression analysis was performed which identified H3K27Me3 level as an independent favorable prognostic factor in all (HR=0.74, 95%CI=0.57-0.95, p=0.02) as well as in P53 mutated AML (n=54, HR=0.48, 95%CI=0.26-0.87, p=0.02). A total of 78 AML patients had molecular data available for the major methylation affecting genes, i.e. IDH1, IDH2, DNMT3A and TET2. The level of H3K27Me3 was not prognostic in patients without any DNA methylation affecting mutation present, but patients with at least one mutation in any of these had better outcome when H3K27Me3 levels were high (highest tertile, figure 1A) compared to those with lower levels (median OS 7.1 vs. 24.1 months, HR=0.42, 95% CI=0.21-0.83, p=0.01, figure 1B). Mutations in U2AF1 and SRSF2 affect the spliceosome and are frequently found in antecedent hematological disorders (AHD), as well as are mutations in chromatin regulating genes ASXL1 and BCOR. We observed significant decreased H3K27Me3 in patients with these mutations corresponding with observed lower levels of H3K27Me3 in patients with AHD than those without (p=0.035). BCOR, SRSF2, U2AF1 and ASXL1 mutations confer poor prognosis in myeloid malignancies, however, in our cohort of 65 sequenced AML patients; not individual or a combination of these mutations were independent prognostic factors, but the degree of H3K27Me3 in these patients (HR= 0.49, 95% CI=0.25-0.95, p=0.03). To recognize dysregulated pathways in AML patients with the identified loss of H3K27Me3, we examined correlations of H3K27Me3 with the other 229 proteins on the array. H3K27Me3 is catalyzed by the polycomb group protein EZH2 and is linked to transcriptional repression via the formation of heterochromatin regions. To identify upregulated proteins and pathways upon the loss of H3K27Me3, we focused on significant negatively correlated proteins with H3K27Me3 leading us to the identification of 20 total and 6 phospho-proteins that showed increased expression upon decreased H3K27Me3. Functional enrichment analysis of this protein set revealed an upregulated anti-apoptotic phenotype. Conclusion:This study shows that proteomic profiling of epigenetic modifications on the histone level have clinical implications in AML and support the idea that epigenetic patterns contribute to a more accurate picture of the leukemic state complementing cytogenetic and molecular genetic subgrouping. Figure 1. A) Lower H3K27Me3 in BM and PB derived AML samples compared to normal CD34+. **** represents p<0.0001, ns = not significant. B) Overall survival probability in AML patients with any DNA methylation affecting mutation (i.e. IDH1/2, DNMT3A, TET2, n=53) according to H3K27Me3 low (blue) and high (orange) status. Figure 1 Disclosures No relevant conflicts of interest to declare.

Chemical Proteomic Profiling of Protein 4′‐Phosphopantetheinylation in Mammalian Cells

AbstractProtein 4′‐phosphopantetheinylation is an essential post‐translational modification (PTM) in prokaryotes and eukaryotes. So far, only five protein substrates of this specific PTM have been discovered in mammalian cells. These proteins are known to perform important functions, including fatty acid biosynthesis and folate metabolism, as well as β‐alanine activation. To explore existing and new substrates of 4′‐phosphopantetheinylation in mammalian proteomes, we designed and synthesized a series of new pantetheine analogue probes, enabling effective metabolic labelling of 4′‐phosphopantetheinylated proteins in HepG2 cells. In combination with a quantitative chemical proteomic platform, we enriched and identified all the currently known 4′‐phosphopantetheinylated proteins with high confidence, and unambiguously determined their exact sites of modification. More encouragingly, we discovered, using targeted chemical proteomics, a potential 4′‐phosphopantetheinylation site in the protein of mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).

Chemical Proteomic Profiling of Protein 4'-Phosphopantetheinylation in Mammalian Cells.

Protein 4'-phosphopantetheinylation is an essential post-translational modification (PTM) in prokaryotes and eukaryotes. So far, only five protein substrates of this specific PTM have been discovered in mammalian cells. These proteins are known to perform important functions, including fatty acid biosynthesis and folate metabolism, as well as β-alanine activation. To explore existing and new substrates of 4'-phosphopantetheinylation in mammalian proteomes, we designed and synthesized a series of new pantetheine analogue probes, enabling effective metabolic labelling of 4'-phosphopantetheinylated proteins in HepG2 cells. In combination with a quantitative chemical proteomic platform, we enriched and identified all the currently known 4'-phosphopantetheinylated proteins with high confidence, and unambiguously determined their exact sites of modification. More encouragingly, we discovered, using targeted chemical proteomics, a potential 4'-phosphopantetheinylation site in the protein of mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).

Quantitative Proteomics for Monitoring Renal Transplant Injury.

This study is aimed at developing a molecular diagnostics platform to enhance the interpretation of renal allograft biopsies using quantitative proteomic profiling of formalin-fixed and paraffin-embedded (FFPE) specimens. A quantitative proteomics platform composed of 1) an optimized FFPE protein sample preparation method, 2) a tandem mass tag TMT10-plex-based proteomic workflow, and 3) a systematic statistical analysis pipeline to reveal differentially expressed proteins has been developed. This platform is then tested on a small sample set (five samples per phenotype) to reveal proteomic signatures that can differentiate T-cell mediated rejection (TCMR) and polyomavirus BK nephropathy (BKPyVN) from healthy functionally stable kidney tissue (STA). Among 2798 quantified proteins, the expression levels of 740 BKPyVN and 638 TCMR associated proteins are significantly changed compared to STA specimens. Principal component analysis demonstrated good segregation of all three phenotypes investigated. Protein detection and quantitation are highly reproducible: replicate comparative analyses demonstrated 71-84% overlap of detected proteins, and the coefficient of variation for protein measurements is <15% in triplicate liquid chromatography-tandem mass spectrometry runs. Quantitative proteomics can be applied to archived FFPE specimens to differentiate different causes of renal allograft injury.

Quantitative In Vivo Proteomics of Metformin Response in Liver Reveals AMPK-Dependent and -Independent Signaling Networks

Metformin is the front-line treatment for type 2 diabetes worldwide. It acts via effects on glucose and lipid metabolism in metabolic tissues, leading to enhanced insulin sensitivity. Despite significant effort, the molecular basis for metformin response remains poorly understood, with a limited number of specific biochemical pathways studied to date. To broaden our understanding of hepatic metformin response, we combine phospho-protein enrichment in tissue from genetically engineered mice with a quantitative proteomics platform to enable the discovery and quantification of basophilic kinase substrates invivo. We define proteins whose binding to 14-3-3 are acutely regulated by metformin treatment and/or loss of the serine/threonine kinase, LKB1. Inducible binding of 250 proteins following metformin treatment is observed, 44% of which proteins bind in a manner requiring LKB1. Beyond AMPK, metformin activates protein kinase D and MAPKAPK2 in an LKB1-independent manner, revealing additional kinases that may mediate aspects of metformin response. Deeper analysis uncovered substrates of AMPK in endocytosis and calcium homeostasis.

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Breaking the ice: urine proteomics of medullary sponge kidney disease

Urinary proteomics is a promising tool for biomarker investigation, particularly in complex kidney diseases. Fabris and colleagues report that urinary laminin subunit alpha-2 is a potential diagnostic marker of medullary sponge kidney (MSK) disease by using a label-free quantitative proteomics platform and a clinically compatible enzyme-linked immunosorbent assay. The neglected issue of stone pathogenesis was also evidenced. This commentary discusses several considerations in biomarker validation, and how urinary proteomics breaks new ground in MSK research.

SWATH-MS Quantitative Proteomic Investigation Reveals a Role of Jasmonic Acid during Lead Response in Arabidopsis.

Lead (Pb) pollution is a growing environment problem that continuously threatens the productivity of crops. To understand the molecular mechanisms of plant adaptation to Pb toxicity, we examined proteome changes in Arabidopsis seedlings following Pb treatment by SWATH-MS, a label-free quantitative proteomic platform. We identified and quantified the expression of 1719 proteins in water- and Pb-treated plants. Among them, 231 proteins showed significant abundance changes (151 elevated and 80 reduced) upon Pb exposure. Functional categorization revealed that most of the Pb-responsive proteins are involved in different metabolic processes. For example, down-regulation of photosynthesis and biosynthesis of isoprenoids and tetrapyrroles in chloroplasts were observed. On the contrary, pathways leading to glutathione, jasmonic acid (JA), glucosinolate (GSL), and phenylpropanoid production are up-regulated. Experimental characterizations demonstrated a rapid elevation of endogenic JA production in Pb-treated Arabidopsis seedlings, while a JA-deficient mutant and a JA-insensitive mutant showed hypersensitivity to root inhibition by Pb, implicating an essential role of JA during Pb responses. Consistently, methyl jasmonate supplementation alleviated Pb toxicity in the wild-type and JA-deficient mutant. Furthermore, GSL levels were substantially enhanced following Pb treatment, while such induction was not detected in the JA mutant, suggesting that the Pb-induced GSL accumulation is JA-dependent. Overall, our work represents the first SWATH-MS analysis in Arabidopsis and highlights a potential mediating role of JA during Pb stress.

LC-MS analysis of Hep-2 and Hek-293 cell lines treated with Brazilian red propolis reveals differences in protein expression.

Red propolis, an exclusive variety of propolis found in the northeast of Brazil has shown to present antitumour activity, among several other biological properties. This article aimed to help to evaluate the underlying molecular mechanisms of the potential anticancer effects of red propolis on tumour, Hep-2, and non-tumour cells, Hek-293. Differentially expressed proteins in human cell lines were identified through label-free quantitative MS-based proteomic platform, and cells were stained with Giemsa to show morphological changes. A total of 1336 and 773 proteins were identified for Hep-2 and Hek-293, respectively. Among the proteins here identified, 16 were regulated in the Hep-2 cell line and 04 proteins in the Hek-293 line. Over a total of 2000 proteins were identified under MS analysis, and approximately 1% presented differential expression patterns. The GO annotation using Protein Analysis THrough Evolutionary Relationships classification system revealed predominant molecular function of catalytic activity, and among the biological processes, the most prominent was associated to cell metabolism. The proteomic profile here presented should help to elucidate further molecular mechanisms involved in inhibition of cancer cell proliferation by red propolis, which remain unclear to date.

Secreted IGFBP5 mediates mTORC1-dependent feedback inhibition of IGF-1 signalling.

The PI(3)K-Akt-mTORC1 pathway is a highly dynamic network that is balanced and stabilized by a number of feedback inhibition loops. Specifically, activation of mTORC1 has been shown to lead to the inhibition of its upstream growth factor signalling. Activation of the growth factor receptors is triggered by the binding of their cognate ligands in the extracellular space. However, whether secreted proteins contribute to the mTORC1-dependent feedback loops remains unclear. We found that cells with hyperactive mTORC1 secrete a protein that potently inhibits the function of IGF-1. Using a large-scale, unbiased quantitative proteomic platform, we comprehensively characterized the rapamycin-sensitive secretome in TSC2(-/-) mouse embryonic fibroblasts, and identified IGFBP5 as a secreted, mTORC1 downstream effector protein. IGFBP5 is a direct transcriptional target of HIF1, which itself is a known mTORC1 target. IGFBP5 is a potent inhibitor of both the signalling and functional outputs of IGF-1. Once secreted, IGFBP5 cooperates with intracellular branches of the feedback mechanisms to block the activation of IGF-1 signalling. Finally, IGFBP5 is a potential tumour suppressor, and the proliferation of IGFBP5-mutated cancer cells is selectively blocked by IGF-1R inhibitors.

Proteomic stratification of clear cell renal cell carcinoma utilizing The Cancer Genome Atlas (TCGA) with external validation.

406 Background: Proteomics represents the ultimate convergence of DNA and expression alterations. We therefore sought to leverage TCGA reverse phase protein array (RPPA) data with an independent proteomic platform to identify druggable targets and pathways associated with prognosis in clear cell renal cell carcinoma (ccRCC). Methods: Unsupervised hierarchical consensus clustering was performed and differentially expressed proteins were identified for pathway analysis. Associations with clinicogenomic factors were assessed and Cox proportional hazards models were performed for disease-specific survival (DSS). Results: RPPA clustering of 324 patients from the ccRCC TCGA revealed 5 robust clusters characterized by alterations in specific pathways and divergent prognoses. Cluster 1 was characterized by poor DSS, decreased expression of receptor tyrosine kinases (RTK) and upregulation of the mTOR pathway. It was also associated with mTOR pathway genomic alterations, sarcomatoid histology and the ccb prognostic mRNA signature (all p&lt;0.001). Cluster 2 was characterized by increased expression of RTKs and interestingly, had upregulation of the mTOR pathway with excellent DSS. After accounting for stage and grade, cluster designation remained independently associated with DSS (HR 0.23 for cluster 2, 95% CI 0.08-0.68; p=0.008). External validation was performed on a separate cohort of 189 patients with a different quantitative proteomics platform. A panel of phosphoproteins (pHER1, pHER2, pHER3, pSHC, pMEK, pAKT), highly discriminant between the most divergent RPPA clusters (1 and 2) was evaluated. Those at the highest quartile of activation in &gt; 3 proteins were associated with improved DSS (HR 0.19, 95% CI 0.05-0.082; p=0.03). Patients with mTOR pathway activation segregated to those with coincident RTK activation (n=83) and those without (n=13). Conclusions: We have identified and validated proteomic signatures which cluster ccRCC patients into 5 prognostic groups. Furthermore, two distinct mTOR-activated clusters—one with high RTK activity and one with increased mTOR pathway genomic alterations were revealed, which may have prognostic and therapeutic implications.

A versatile reversed phase-strong cation exchange-reversed phase (RP-SCX-RP) multidimensional liquid chromatography platform for qualitative and quantitative shotgun proteomics.

An automatable, robust, high-performance online multidimensional liquid chromatography (MDLC) platform comprising of pH 10 reversed-phase (RP), strong cation exchange (SCX), and pH 2 RP separation stages has been integrated into a modified commercial off-the-shelf LC instrument with a simple rewiring, enabling accelerated routine qualitative and quantitative proteomics analyses. This system has been redesigned with a dual-trap column configuration to improve the throughput by greatly decreasing the system idle time. The performance of this new design has been benchmarked through analysis of the total lysate of S. cerevisiae, in comparison with that of the former tailor-made system featuring more complicated components; the total run time per "load-and-go" LC/MS analysis was approximately 24 h, with minimal idle time and no labor-intensive steps. This platform features high-resolution fractionations, ease of use and a high degree of user programmability in the first two chromatographic dimensions, allowing flexible and effective sampling with (RP-SCX-RP) or without (RP-RP) the inclusion of SCX sub-fractionation; good proteome coverage and reproducibility was demonstrated through the analyses of bacterial, cell culture, and monkey brain tissue proteomes. The viability of the 3D RP-SCX-RP has been proven in proteome-wide studies of STO fibroblasts and yeast tryptic digests, resulting in extended proteome and protein coverages with high reproducibility-in particular, discovering extra-hydrophilic peptides-at the expense of the acquisition time. The identified inventory of the rat pheochromocytoma PC12 cell proteome-a total of 6345 proteins and 97 309 unique peptides is the most comprehensive dataset to date-provides an example of the value of the 3D RP-SCX-RP. The use of orthogonal chromatographic dimensions in the 3D RP-SCX-RP also circumvents the issues of isobaric interference of mass-tagging background contaminations, while significantly improving the accuracy of isobaric tags for relative and absolute quantitation (iTRAQ)-based protein quantitation experiments.