Application of 2‐D free‐flow electrophoresis/RP‐HPLC for proteomic analysis of human plasma depleted of multi high‐abundance proteins

Free-flow electrophoresis (FFE) and rapid (6 min) RP-HPLC was used to fractionate human citrate-treated plasma. Prior to analysis, the six most abundant proteins in plasma were removed by immunoaffinity chromatography; both depleted plasma and the fraction containing the six abundant proteins depleted were taken for MS-based analysis. Fractionated proteins were digested with trypsin and the generated peptides were subjected to MS-based peptide sequencing. To date, 78 plasma proteins have been unambiguously identified by manual validation from 16% (15/96 FFE total fractions) of the collected FFE pools; 55 identifications were based on > or = 2 tryptic peptides and 23 using single peptides. The molecular weight range of proteins and peptides isolated by this method ranged from approximately 190 K (e.g., Complement C3 and C4) to approximately 4-6 K (e.g., CRISPP and Apolipoprotein C1). This FFE/RP-HPLC approach reveals low-abundance proteins and peptides (e.g., L-Selectin approximately 17 ng/mL and the cancer-associated SCM-recognition, immunodefense suppression, and serine protease protection peptide (CRISPP) at approximately 0.5-1 ng/mL), where CRISPP was found in association with alpha-1-antitrypsin as a non-covalent complex, in the fraction containing the depleted high-abundance proteins. In contrast to shotgun proteomic approaches, the FFE/RP-HPLC method described here allows the identification of potentially interesting peptides to be traced back to their protein of origin, and for the first time, has confirmed the "protein sponge" hypothesis where the 35 residue CRISPP polypeptide is non-covalently complexed with the major circulating plasma protein alpha-1-antitrypsin.