Normal human plasma proteome by 2DLC label-free approach
Abstract
Body fluid is considered a rich source of disease biomarkers. Proteins in plasma have potential clinical applications for disease diagnostic and prognostic predictions. To determine the protein components and functional features, a proteomic analysis of plasma was conducted by high-resolution mass spectrometry. As a result, a total of 1189 proteins were identified, and the concentrations of 424 proteins were estimated by an intensity-based algorithm quantitation method. The plasma-enriched proteins were mainly involved in the hematological system, immune system, complement system, and integrin signaling pathway.
In this study, we conducted a proteomic analysis using high-resolution mass spectrometry. For plasma, the relative protein abundances and average protein concentrations were estimated using intensity-based algorithm quantitation (iBAQ) methods.
All samples were obtained from normal healthy individuals. The volunteers were examined by physicians at the Peking Union Medical College Hospital .
| Protocol | Description |
|---|---|
| Clinical materials |
Plasma is obtained from the Peking Union Medical College Hospital. |
| Sample preparation |
Pooled plasma samples were centrifuged to remove precipitates. The pooled plasma were depleted of 14 high-abundance proteins by using a Human 14 affinity LC column. The proteins were digested using filter-aided sample preparation methods.Briefly, the samples were denatured with 50 mM dithiothreitol, alkylated in 55 mM iodoacetamide, and digested with trypsin (1:50) at 37 °C overnight. |
| iTRAQ Or TMT labeling | |
| HPLC |
After digestion, the lyophilized samples were fractionated by offline high pH reversed-phase liquid chromatography (RPLC) columns (4.6mm×250mm,C18, 3μm;Waters Corp,Milford,MA, USA). |
| LC/MS/MS |
Each sample was analyzed on a reverse-phase C18 self-packed capillary LC column (75 μm×100mm,3μm). A Triple TOF 5600 system was used to analyze the sample, and the MS data were acquired in a high-sensitivity mode using the following parameters: 30 data-dependent MS/MS scans per full scan; full scans were acquired at a resolution of 40 000, and MS/MS scans were acquired at a resolution of 20 000; rolling collision energy; charge state screening (including precursors with +2 to +4 charge states); dynamic exclusion (exclusion duration 15 s); MS/MS scan range of 250–1800 m/z; and a scan time of 50 ms. |
| Data processing |
The MS/MS data were processed using the Mascot software (version 2.3.02, Matrix Science, London, UK) and searched against the Swiss-Prot database (Homo sapiens, 20 267 sequences, 2013 07 version). The Mascot search results were filtered using the decoy database method in Scaffold (version 4.3.2, Proteome Software Inc., Portland, OR, USA). |