Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery.
The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n>250) sequenced in these profiles came from a surprisingly small number of proteins (n approximately 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.
Koomen
John M
JM
Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, 0089, UT M.D., 1515 Holcombe Blvd., Houston, Texas 77030, USA.
Li
Donghui
D
Xiao
Lian-chun
LC
Liu
Thomas C
TC
Coombes
Kevin R
KR
Abbruzzese
James
J
Kobayashi
Ryuji
R
D015415:Biomarkers / Q000097:blood; D001777:Blood Coagulation; D001798:Blood Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis; D016022:Case-Control Studies; D003167:Complement Activation; D006801:Humans; D010190:Pancreatic Neoplasms / Q000097:blood; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015415:Biomarkers; D001798:Blood Proteins; D002352:Carrier Proteins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D010447:Peptide Hydrolases