Additional file 1: of PGA: an R/Bioconductor package for identification of novel peptides using a customized database derived from RNA-Seq

Supporting methods. (DOCX 27 kb

Expansion of the Ion Library for Mining SWATH-MS Data through Fractionation Proteomics

The strategy of sequential window acquisition of all theoretical fragment ion spectra (SWATH) is emerging in the field of label-free proteomics. A critical consideration for the processing of SWATH data is the quality of the ion library (or mass spectrometric reference map). As the availability of open spectral libraries that can be used to process SWATH data is limited, most users currently create their libraries in-house. Herein, we propose an approach to construct an expanded ion library using the data-dependent acquisition (DDA) data generated by fractionation proteomics. We identified three critical elements for achieving a satisfactory ion library during the iterative process of our ion library expansion, including a correction of the retention times (RTs) gained from fractionation proteomics, appropriate integrations of the fractionated proteomics into an ion library, and assessments of the impact of the expanded ion libraries to data mining in SWATH. Using a bacterial lysate as an evaluation material, we employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate the lysate proteins and constructed the expanded ion library using the fractionation proteomics data. Compared with the ion library built from the unfractionated proteomics, approximately 20% more peptides were extracted from the expanded ion library. The extracted peptides, moreover, were acceptable for further quantitative analysis

Digging More Missing Proteins Using an Enrichment Approach with ProteoMiner

Human Proteome Project (HPP) aims at mapping entire human proteins with a systematic effort upon all the emerging techniques, which would enhance understanding of human biology and lay a foundation for development of medical applications. Until now, 2563 missing proteins (MPs, PE2–4) are still undetected even using the most sensitive approach of protein detection. Herein, we propose that enrichment of low-abundance proteins benefits MPs finding. ProteoMiner is an equalizing technique by reducing high-abundance proteins and enriching low-abundance proteins in biological liquids. With triton X-100/TBS buffer extraction, ProteoMiner enrichment, and peptide fractionation, 20 MPs (at least two non-nested unique peptides with more than eight a.a. length) with 60 unique peptides were identified from four human tissues including eight membrane/secreted proteins and five nucleus proteins. Then 15 of them were confirmed with two non-nested unique peptides (≥9 a.a.) identified by matching well with their chemically synthetic peptides in PRM assay. Hence, these results demonstrated ProteoMiner as a powerful means in discovery of MPs

Identification of Novel Biomarkers for Sepsis Prognosis via Urinary Proteomic Analysis Using iTRAQ Labeling and 2D-LC-MS/MS

<div><h3>Objectives</h3><p>Sepsis is the major cause of death for critically ill patients. Recent progress in proteomics permits a thorough characterization of the mechanisms associated with critical illness. The purpose of this study was to screen potential biomarkers for early prognostic assessment of patients with sepsis.</p> <h3>Methods</h3><p>For the discovery stage, 30 sepsis patients with different prognoses were selected. Urinary proteins were identified using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS. Mass spec instrument analysis were performed with Mascot software and the International Protein Index (IPI); bioinformatic analyses were used by the algorithm of set and the Gene Ontology (GO) Database. For the verification stage, the study involved another 54 sepsis-hospitalized patients, with equal numbers of patients in survivor and non-survivor groups based on 28-day survival. Differentially expressed proteins were verified by Western Blot.</p> <h3>Results</h3><p>A total of 232 unique proteins were identified. Proteins that were differentially expressed were further analyzed based on the pathophysiology of sepsis and biomathematics. For sepsis prognosis, five proteins were significantly up-regulated: selenium binding protein-1, heparan sulfate proteoglycan-2, alpha-1-B glycoprotein, haptoglobin, and lipocalin; two proteins were significantly down-regulated: lysosome-associated membrane proteins-1 and dipeptidyl peptidase-4. Based on gene ontology clustering, these proteins were associated with the biological processes of lipid homeostasis, cartilage development, iron ion transport, and certain metabolic processes. Urinary LAMP-1 was down-regulated, consistent with the Western Blot validation.</p> <h3>Conclusion</h3><p>This study provides the proteomic analysis of urine to identify prognostic biomarkers of sepsis. The seven identified proteins provide insight into the mechanism of sepsis. Low urinary LAMP-1 levels may be useful for early prognostic assessment of sepsis.</p> <h3>Trial Registration</h3><p>ClinicalTrial.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT01493492">NCT01493492</a></p> </div

GO annotation of the final selected differentially expressed proteins.

<p>These differentially expressed proteins were classified among three categories: cellular component (CC), molecular function (MF) and biological process (BP). According to the GO database, the top 10 components for CC, MF, BP of the selected differentially expressed proteins are shown along with their enrichment score, represented as a <i>p</i>-value.</p

Demographics of subjects in the discovery and verification stages.

<p>Quantitative data of normal distribution are presented as mean±SD. Quantitative data of non-normal distribution are presented as median (interquartile range). Qualitative data are presented as n(%).</p><p>WBC counts, white blood cell counts; CRP, C-reactive protein; PCT, Procalcitionin; APACHE II score, Acute Physiologic Assessment and Chronic Health Evaluation II scores; SOFA score, Sequential Organ Failure Assessment scores.</p

Western blot validation of three candidate markers in individual sepsis patients with different prognoses.

<p>(A) Relative protein expression of SBP-1. The survivor group and non-survivor groups were 0.938±0.347 and 0.945±0.602 (<i>p</i>>0.05), respectively. (B) Relative protein expression of LAMP-1. The survivor group and non-survivor groups were 0.752±0.246 and 0.617±0.166 (<i>p</i><0.05), respectively. (C) Relative protein expression of HSPG-2. The survivor and non-survivor groups were 0.802±0.282 and 0.880±0.606 (<i>p</i>>0.05), respectively.</p

Proteins identified in the three sets (technical replicates).

<p>Proteins identified in the three sets (technical replicates).</p

Appraisal of the Missing Proteins Based on the mRNAs Bound to Ribosomes

Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins. Total of 1501 missing proteins were found by neither RNC-mRNA nor MS/MS in the three liver cancer cell lines. For these missing proteins, some are expected higher hydrophobicity, unsuitable detection, or sensory functions as properties at the protein level, while some are predicted to have nonexpressing chromatin structures on the corresponding gene level. With further integrated analysis, we could attribute 93% of them (1391/1501) to these causal factors, which result in the expression products scarcely detected by RNA-seq or MS/MS

Summary of differentially expressed proteins identified in SP/SI, de/SI and de/SP among the three sets.

<p>SI: urine specimens from patients with SIRS.</p><p>SP: urine specimens from sepsis patients, acquired within 24 h of admission to the ICU.</p><p>de: urine specimens from sepsis patients, acquired within 48 h before death.</p><p>de/SI: proteins differentially expressed in de relative to SI.</p><p>de/SP: proteins differentially expressed in de relative to SP.</p><p>SP/SI: proteins differentially expressed in SP relative to SI.</p