Comparison of methods to examine the endogenous peptides of fetal calf serum |
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Authors: | Declan Williams Peihong Zhu Peter Bowden Catherine Stacey Mike McDonell Paul Kowalski Jane Marie Kowalski Ken Evans Eleftherios P Diamandis K W Michael Siu John Marshall |
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Institution: | (1) Bruker Daltonics, Inc., Bellerica, MA;(2) Department of Pathology and Laboratory Medicine, Mount Sinai Hospital and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada;(3) Department of Chemistry and Biology, Faculty of Engineering and Applied Science, Ryerson University, 350 Victoria Street, M5B 2K3 Toronto, ON, Canada;(4) Ontario Cancer Biomarker Network, MaRS, 101 College Street, Toronto Ontario, ON, Canada |
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Abstract: | There is a great desire to relate the patterns of endogenous peptides in blood to human disease and drug response. The best
practices for the preparation of blood fluids for analysis are not clear and also relatively few of the peptides in blood
have been identified by tandem mass spectrometry. We compared a number of sample preparation methods to extract endogenous
peptides including C18 reversed phase, precipitation, and ultrafiltration. We examined the results of these sample preparation
methods by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-tandem mass spectrometry
(MS/MS) using MALDI-TOF/TOF and electrospray ionization-ion trap. Peptides from solid-phase extraction with C18 in the range
of hundreds of femtomoles per spot were detected from the equivalent of 1 μL of serum by MALDI-TOF. We observed endogenous
serum peptides from fibrinogen α- and β-chain, complement C3, α-2-HS-glycoprotein, albumin, serine (or cysteine) proteinase
inhibitor, factor VIII, plasminogen, immunoglobulin, and other abundant blood proteins. However, we also recorded significant
MS/MS spectra from tumor necrosis factor-α-, major histocompatibility complex-, and angiotensin-related peptides, as well
as peptides from collagens and other low-abundance proteins. Amino acid substitutions were detected and a phosphorylated peptide
was also observed. This is the first time the endogenous peptides of fetal serum have been examined by MS and where peptides
from low-abundance proteins, phosphopeptides, and amino acid substitutions were detected. |
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Keywords: | Fetal calf serum liquid chromatography-electrospray ionization mass spectrometry matrix-assisted laser desorption/ionization time-of-flight MALDI ESI tandem mass spectrometry |
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