Number of aligned sequences between two databases based different alignment length threshold.

<p>The upper is the aligned sequences number of clementine database, the downer is the aligned sequences number of orange database.</p

Identification comparison between homologous database and transcriptome based database (T. based database).

<p>(A) Venn chart for distribution of the proteins identified by MaxQuant based on two databases. (B) Numbers of proteins identification based on homologous and integrated database.</p

Workflow for identifying peptides based on integrated database.

<p>Workflow for identifying peptides based on integrated database.</p

Distribution (by number and percentage) of protein groups identified with homologous database and integrated database into relevant secondary level GO classes, based on GO annotation.

<p>Distribution (by number and percentage) of protein groups identified with homologous database and integrated database into relevant secondary level GO classes, based on GO annotation.</p

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

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

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

Proteins identified in the three sets (technical replicates).

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

Schematic of the experimental design based on iTRAQ labeling combined with 2-D LC-MS/MS analysis of SI, SP, and de.

<p>SCX, strong cation exchange. SI: urine specimens from patients with SIRS. SP: urine specimens from sepsis patients, acquired within 24 h of admission to the ICU. de: urine specimens from sepsis patients, acquired within 48 h before death.</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