A novel approach and protocol for discovering extremely low-abundance proteins in serum

Proteomics. 2006 Sep;6(17):4845-55. doi: 10.1002/pmic.200500774.

Abstract

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.

MeSH terms

  • Blood Proteins / analysis*
  • Blood Proteins / chemistry
  • Blood Proteins / isolation & purification
  • Blood Proteins / metabolism
  • Chromatography, Liquid
  • Humans
  • Mass Spectrometry
  • Molecular Weight
  • Sensitivity and Specificity

Substances

  • Blood Proteins