Experimental schema of the discovery and verification phases.

<p>Left panel, serum samples collection and depletion to remove the human serum albumin (HSA); Middle panel, IMDL fractionation which separates serum peptides by their pIs (2.5∼8.5) and hydrophobicity, with an example of HPLC-MS/MS chromatography showed for each pH step elution; Right panel, the number of NSCLC patients and the number of age-matched healthy controls in the verification phase by western blotting, TMA and MRM measurement. SCX, strong cation exchanger, RP reversed-phase chromatography. AD: Adenocarcinoma; SCC: Squamous cell carcinoma; NSCLC: Non-small cell lung cancer.</p

A high quality serum proteome associated with NSCLC.

<p>(A) Hierarchical clustering heatmap of 101 significantly altered proteins. Expression values are shown as a color scale with higher values represented by yellow and lower represented by blue. (B) Gene Ontology biological processes significantly enriched in 101 protein set. Gene symbols in blue or red circles (could be referred in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051748#pone.0051748.s003" target="_blank">Table S3</a>) indicate the corresponding proteins in down- or up-regulated in NSCLC sera.</p

Workflow of western blotting assisted verification for A1BG.

<p>Western blot images across 12 ADs, 12 SCCs and 12 control sera. Coomassie stained Albumin (ALB) was shown as the loading controls. IOD values were extracted and normalized by this calibration sample, suggesting the differential distribution of A1BG serum levels between normal and lung cancer cases.</p

Serum proteins potentially relevant to NSCLC confirmed by IMDL and label free quantification in the discovery phase.

a<p>Averaged PSMs (peptide hits) in Normal (N), AD and SCC group.</p>b<p>Reference listed were closely related to NSCLC and all reported the identical up or down regulation trend with our results.</p

HCA and PCA of total serum proteomic data between samples.

<p>(A) Hierarchical clustering analysis and (B) Principal component analysis of all the 647 proteins quantified across the 18 serum samples. Ends of red arrows in (B) represent the PCA results for each protein.</p

TMA assisted validation for histological biomarkers.

<p>(A) Representative IHC cases with higher expression level in NSCLC sections. One SCC and one AD sample are shown to suggest different Q scores can be obtained by the four proteins; magnification 200×. (B) Virtual result of TMA. Q scores are calculated considering both percentage of positive cells and stain intensity and shown as a color scale with higher Q represented by yellow (maximum = 27) and lower Q represented by blue (minimum = 0). The grey spot represents the only one case lost during the processing.</p

Multiplexed Targeted Mass Spectrometry-Based Assays for the Quantification of N‑Linked Glycosite-Containing Peptides in Serum

Protein glycosylation is one of the most common protein modifications, and the quantitative analysis of glycoproteins has the potential to reveal biological functions and their association with disease. However, the high throughput accurate quantification of glycoproteins is technically challenging due to the scarcity of robust assays to detect and quantify glycoproteins. Here we describe the development of multiplexed targeted MS assays to quantify N-linked glycosite-containing peptides in serum using parallel reaction monitoring (PRM). Each assay was characterized by its performance metrics and criteria established by the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (NCI CPTAC) to facilitate the widespread adoption of the assays in studies designed to confidently detect changes in the relative abundance of these analytes. An in-house developed software program, MRMPlus, was used to compute assay performance parameters including specificity, precision, and repeatability. We show that 43 selected N-linked glycosite-containing peptides identified in prostate cancer tissue studies carried out in our group were detected in the sera of prostate cancer patients within the quantitative range of the developed PRM assays. A total of 41 of these formerly N-linked glycosite-containing peptides (corresponding to 37 proteins) were reproducibly quantified based on their relative peak area ratios in human serum during PRM assay development, with 4 proteins showing differential significance in serum from nonaggressive (NAG) vs aggressive (AG) prostate cancer patient serum (<i>n</i> = 50, NAG vs AG). The data demonstrate that the assays can be used for the high throughput and reproducible quantification of a panel of formerly N-linked glycosite-containing peptides. The developed assays can also be used for the quantification of formerly N-linked glycosite-containing peptides in human serum irrespective of disease state

Data_Sheet_1_Prevalence and effect on prognosis of sarcopenia in patients with primary biliary cholangitis.docx

BackgroundSarcopenia adversely affects the treatment outcomes in Cirrhosis and NAFLD. However, such research is limited in primary biliary cholangitis (PBC) patients. This study was performed to examine the prevalence of sarcopenia and its impact on PBC patients’ prognoses.MethodsThis study enrolled confirmed PBC patients who had an abdominal CT scan. Sarcopenia was determined by the L3-skeletal muscle index with a Chinese population-based cut-off value. Laboratory test values and liver stiffness measurements values were obtained from the electronic medical records.ResultsIn total, 174 PBC patients with a median age of 54 (IQR, 48, 62) years old, were enrolled. 45 (25.9%) patients among them were diagnosed with sarcopenia. Univariate and multivariate logistic regression results illustrated that male gender (OR = 9.152, 95%CI = 3.131–26.751, p ConclusionThe current findings illustrate that comprehensive evaluation and management of sarcopenia may contribute to the improvement of treatment outcomes and life quality of PBC patients.</p