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Human embryonic stem cells (hESCs) have the capacity for self-renewal and multilineage differentiation, which are of clinical importance for regeneration medicine. Despite the significant progress of hESC study, the complete hESC proteome atlas, especially the surface protein composition, awaits delineation. According to the latest release of neXtProt database (January 17, 2018; 19 658 PE1, 2, 3, and 4 human proteins), membrane proteins present the major category (1047; 48%) among all 2186 missing proteins (MPs). We conducted a deep subcellular proteomics analysis of hESCs to identify the nuclear, cytoplasmic, and membrane proteins in hESCs and to mine missing membrane proteins in the very early cell status. To our knowledge, our study achieved the largest data set with confident identification of 11 970 unique proteins (1% false discovery rate at peptide, protein, and PSM levels), including the most-comprehensive description of 6 138 annotated membrane proteins in hESCs. Following the HPP guideline, we identified 26 gold (neXtProt PE2, 3, and 4 MPs) and 87 silver (potential MP candidates with a single unique peptide detected) MPs, of which 69 were membrane proteins, and the expression of 21 gold MPs was further verified either by multiple reaction monitoring mass spectrometry or by matching synthetic peptides in the Peptide Atlas database. Functional analysis of the MPs revealed their potential roles in the pluripotency-related pathways and the lineage- and tissue-specific differentiation processes. Our proteome map of hESCs may provide a rich resource not only for the identification of MPs in the human proteome but also for the investigation on self-renewal and differentiation of hESC. All mass spectrometry data were deposited in ProteomeXchange via jPOST with identifier PXD009840.

Introduction: Human embryonic stem cells (hESCs) have unique biological features and attributes that make them attractive in various areas of biomedical research. With heightened applications, there is an ever increasing need for advancement of proteome analysis. Membrane proteins are one of the most important subset of hESC proteins as they can be used as surface markers. Areas covered: This review discusses commonly used surface markers of hESCs, and provides in-depth analysis of available hESC membrane proteome reports and the existence of these markers in many other cell types, especially cancer cells. Appreciating, existing ambiguity in the definition of a membrane protein, we have attempted a meta analysis of the published membrane protein reports of hESCs by using a combination of protein databases and prediction tools to find the most confident plasma membrane proteins in hESCs. Furthermore, responsiveness of plasma membrane proteins to differentiation has been discussed based on available transcriptome profiling data bank. Expert commentary: Combined transcriptome and membrane proteome analysis highlighted additional proteins that may eventually find utility as new cell surface markers.

Objectives: No study has addressed how the immune status at the molecular level is affected by first-line pemetrexed and cisplatin (PEM-CIS) combination therapy in patients with non-small-cell lung cancer (NSCLC). Thus, we aimed to identify the immune status from membrane proteome alterations in patients with NSCLC upon PEM-CIS treatment., Methods: The paired peripheral blood mononuclear cells (PBMCs) were collected from four patients with lung adenocarcinoma before and after the first regimen of PEM-CIS treatment and applied quantitative membrane proteomics analysis., Result: In the personalized PBMC membrane proteome profiles, 2424 proteins were identified as displaying patient-specific responsive patterns. We discovered an elevated neutrophil activity and a more suppressive T-cell phenotype with the downregulation of cytotoxic T lymphocyte antigen 4 degradation and the upregulation of type 2 T-helper and T-regulatory cells in the patient with the highest progression-free survival (PFS) of 14.5 months. Patients with a PFS of 2 months showed higher expressions of T-cell subsets, MHC class II pathways, and T-cell receptor signaling, which indicated an activated immune status., Conclusion and Clinical Relevance: Without the additional isolation of specific immune cell populations, our study demonstrated that PEM-CIS chemotherapy altered patients' immune system in terms of neutrophils, T cells, and antigen presentation pathways., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Diabetic cardiomyopathy is a well-recognized complication of diabetes, but its pathophysiology is unclear. We aimed to investigate the mechanisms underlying cardiac dysfunction in an elderly type 2 diabetic (T2DM) mouse model, using membrane proteomic analyses. Elderly mice were fed a high fat diet for 12 weeks to induce T2DM, and myocardial structure and function were assessed by echocardiography. Cardiomyocytes were isolated by Langendorff perfusion and subjected to iTRAQ-based quantitative membrane proteomic profiling, immunoblotting, and real-time quantitative reverse-transcriptase polymerase chain reaction. Compared to controls, elderly T2DM mice showed worse systolic function, more myocardial fibrosis and up-regulation of several heart failure markers (all p < 0.05). Cardiomyocyte membrane proteomic profiling revealed that 417 proteins had differential expressions related to perturbations in several biological processes in T2DM mice compared with the control. The most up-regulated proteins were involved in oxidative phosphorylation, whereas many down-regulated proteins were involved in cytoskeletal regulation. Differential protein expression correlated with myocardial systolic velocity by tissue Doppler. In addition, cardiomyocyte immunofluorescence staining showed greater disorganization of thick/parallel F-actin stress fibers and marked reduction in F-to-G-actin ratio in T2DM vs control (p < 0.05), which paralleled worsened myocardial systolic velocity. We concluded that cardiac contractile dysfunction in elderly T2DM mice was associated with impaired energetics and cytoskeletal disorganization.

Background: To identify novel serological biomarkers for human colorectal cancer (CRC), we analyzed CRC tissues using gel-assisted digestion and isobaric tags with related and absolute quantitation (iTRAQ) labeling mass spectrometry (MS). By comparing pairs of tumor tissues and matched normal tissues, we discovered the SEC61β with expression changes 3.3-fold and a marginal statistical significance (p=0.052) previously., Methods: SEC61β expression in CRC tissues was further analyzed by western blotting and immunohistochemistry. We next assessed the putative diagnostic value of the SEC61β autoantibody as a serum marker., Results: Using western blotting analysis, SEC61β expression was increased 1.9-fold in tumor tissues. Immunohistochemical analysis of 64 CRC specimens showed that SEC61β was positively detected in 64% of the tumors, but weakly or not detected in >80% of the adjacent nontumor epithelial cells. Western blot analysis with plasma samples showed that the sensitivity and specificity of the SEC61β autoantibody from patients with CRC were 79% and 75%, respectively. Importantly, the results of the SEC61β autoantibody for early detection of colorectal cancer revealed a higher sensitivity of 77% than the carcinoembryonic antigen (CEA) assay., Conclusions: Measurement of SEC61β autoantibody levels may provide an alternative detection indicator for CRC, particularly among early-stage patients., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48-70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.

The aim of this study was to uncover the membrane protein profile differences between colorectal carcinoma and neighboring normal mucosa from colorectal cancer patients. Information from cellular membrane proteomes can be used not only to study the roles of membrane proteins in fundamental biological processes, but also to discover novel targets for improving the management of colorectal cancer patients. We used solvent extraction and a gel-assisted digestion method, together with isobaric tags with related and absolute quantitation (iTRAQ) reagents to label tumoral and adjacent normal tissues in a pairwise manner (n = 8). For high-throughput quantification, these digested labeled peptides were combined and simultaneously analyzed using LC-MS/MS. Using the shotgun approach, we identified a total of 438 distinct proteins from membrane fractions of all eight patients. After comparing protein expression between cancerous and corresponding normal tissue, we identified 34 upregulated and eight downregulated proteins with expression changes greater than twofold (Student's t-test, P < 0.05). Among these, the overexpression of well-established biomarkers such as carcinoembryonic antigens (CEACAM5, CEACAM6), as well as claudin-3, HLA class I histocompatibility antigen A-1, tapasin and mitochondrial solute carrier family 25A4 were confirmed by western blotting. We conclude that gel-assisted digestion and iTRAQ labeling MS is a potential approach for uncovering and comparing membrane protein profiles of tissue samples that has the potential to identify novel biomarkers.

Toward multiplexed, comprehensive, and robust quantitation of the membrane proteome, we report a strategy combining gel-assisted digestion, iTRAQ (isobaric tags for relative and absolute quantitation) labeling, and LC-MS/MS. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (<8% error) and precision (<12% relative S.D.), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate = 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane proteins, each quantified based on an average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that achieved previously using MS and provides superior quantitation accuracy compared with other methods. We applied this approach to the first proteomics delineation of phenotypic expression in a mouse model of autosomal dominant polycystic kidney disease (ADPKD). By characterizing kidney cell plasma membrane from wild-type versus PKD1 knock-out mice, 791 proteins were quantified, and 67 and 37 proteins showed > or =2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease.