The endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by LC-ESI-Paul ion trap and Qq-TOF.
Many peptides of biological or medicinal importance may be derived from proteolytic actions and are found at low concentrations in human blood fluids. Endogenous polypeptides from human serum were precipitated in acetonitrile and the precipitate was then selectively extracted with water modified by organic solvents and collected over C18 resin. Extraction of serum with C18 alone, and the acetonitrile supernatant or ultrafiltration collected over C18, served as controls. The samples were analyzed by SDS-PAGE, or C18 high pressure liquid chromatography with electrospray ionization using a Paul ion trap and Qq-TOF. Spectra were correlated without specifying an enzyme using the X!TANDEM or the Paragon algorithms. Multiple endogenous peptides from plasminogen, coagulation factors, collagens, serum amyloid, receptors, zinc finger/bromo peptide proteins, ryanodine receptor, calmodulin binding activator, erythroid differentiation factor, testes cancer antigen, extracellular matrix protein, myeloid/lymphoid leukemia 2 and many low abundance proteins were correlated by X!TANDEM with protein expect values of approximately E-16 or less. Proteins with binding sites for nucleic acids, phosphoinositides, and other cellular locations were also observed using the Qq-TOF and Paragon algorithm. Proteins with low expectation scores and overlapping peptides sequences were observed. The existence of these proteins in serum has been confirmed by tryptic digestion and LC-ESI-MS/MS. The presence of plasminogen, serum amyloid and zinc finger RNA binding proteins were confirmed by Western blot. There was agreement on the detection of endogenous peptides from low abundance proteins associated with the biology of cancer from the examination of the blood peptides by ion trap and Qq-TOF, tryptic digests of blood proteins, and Western blot.
Tucholska
Monika
M
Department of Chemistry and Biology, Faculty of Engineering and Applied Science, 350 Victoria Street, Toronto, ON, Canada M5B 2K3.
Florentinus
Angelique
A
Williams
Declan
D
Marshall
John G
JG
D000097:Acetonitriles / Q000737:chemistry*; D000465:Algorithms; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry*; D002845:Chromatography / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007477:Ions; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000097:Acetonitriles; D001798:Blood Proteins; D007477:Ions; D010455:Peptides; D020543:Proteome; C032159:acetonitrile