title: Precipitation and selective extraction of human serum endogenous peptides with analysis by quadrupole time-of-flight mass spectrometry reveals posttranslational modifications and low-abundance peptides.
abstract: The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous blood peptides can be effectively enriched by precipitation of the serum with organic solvents followed by selective extraction of peptides using aqueous solutions modified with organic solvents. Polypeptides on filter paper were assayed with Coomasie brilliant blue binding. The polypeptides were resolved by detergent tricine polyacrylamide electrophoresis and visualized by diamine silver staining. Peptides in the extracts were collected by C18 and analyzed by matrix-assisted laser desorption/ionization and liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) quadrupole time-of-flight MS/MS. Peptides were resolved as multiple isotopic peaks in MS mode with mass deviation of 0.1 Da or less and similar accuracy for fragments. The sensitivity of MS and MS/MS analysis was estimated to be in the picomolar range or less. The peptide composition of the extracts was dependent on solvent formulation. Multiple peptides from apolipoproteins, complement proteins, coagulation factors, and many others were identified by X!Tandem with high mass accuracy of peptide ions and fragments from collision-induced dissociation. Many previously unreported posttranslational modifications of peptides including phosphorylations, oxidations, glycosylations, and others were detected with high mass accuracy and may be of clinical importance. About 4,630 redundant peptides were identified with 99% confidence separately, and together some 1,251 distinct proteins were identified with 99% confidence or greater using the Paragon algorithm.
authors.affiliation: Department of Chemistry and Biology, Faculty of Engineering and Applied Science, Ryerson University, 350 Victoria Street, Toronto, ON, M5B 2K3, Canada.
authors.forename: Declan
authors.initials: D
authors.lastname: Williams
authors.forename: Suzanne
authors.initials: S
authors.lastname: Ackloo
authors.forename: Peihong
authors.initials: P
authors.lastname: Zhu
authors.forename: Peter
authors.initials: P
authors.lastname: Bowden
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Evans
authors.forename: Christina L
authors.initials: CL
authors.lastname: Addison
authors.forename: Chris
authors.initials: C
authors.lastname: Lock
authors.forename: John G
authors.initials: JG
authors.lastname: Marshall
mesh_terms: D000595:Amino Acid Sequence; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000097:blood* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010455:Peptides