Targeted protein quantitation and profiling using PVDF affinity probe and MALDI-TOF MS.
Development of a rapid, effective, and highly specific platform for target identification in complex biofluids is one of the most important tasks in proteomic research. Taking advantage of the natural hydrophobic interaction of PVDF with probe protein, a simple and effective method was developed for protein quantitation and profiling. Using antibody-antigen interactions as a proof-of-concept system, the targeted plasma proteins, serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP), could be selectively isolated and enriched from human plasma by antibody-immobilized PVDF membrane and directly identified by MALDI-TOF MS without additional elution step. The approach was successfully applied to human plasma for rapid quantitation and variant screening of SAP, SAA, and CRP in healthy individuals and patients with gastric cancer. The triplexed on-probe quantitative analysis revealed significant overexpression of CRP and SAA in gastric cancer group, consistent with parallel ELISA measurements and pathological progression and prognostic significance reported in previous literatures. Furthermore, the variant mass profiling of the post-translationally modified forms revealed a high occurrence of de-sialic acid SAP in patients with gastric cancer. Due to the versatile assay design, ease of probe preparation without chemical synthesis, and compatibility with MALDI-TOF MS analysis, the methodology may be useful for target protein characterization, functional proteomics, and screening in clinical proteomics.
Chen
Shu-Hua
SH
Institute of Chemistry, Academia Sinica, Taipei, Taiwan, ROC.
Liao
Hsin-Kai
HK
Chang
Chih-Yang
CY
Juo
Chiun-Gung
CG
Chen
Jenn-Han
JH
Chan
Sunney I
SI
Chen
Yu-Ju
YJ
D002097:C-Reactive Protein / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008567:Membranes, Artificial; D008954:Models, Biological; D011145:Polyvinyls / Q000737:chemistry*; D011379:Prognosis; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D000685:Serum Amyloid A Protein / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013274:Stomach Neoplasms / Q000097:blood / Q000175:diagnosis / Q000378:metabolism / Q000473:pathology; D005465:Fluorocarbon Polymers
D002097:C-Reactive Protein; D008567:Membranes, Artificial; D011145:Polyvinyls; D000685:Serum Amyloid A Protein; D000683:Serum Amyloid P-Component; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers