Targeted protein quantitation and profiling using PVDF affinity probe and MALDI‐TOF MS

Abstract

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.

Discovery and verification of amyotrophic lateral sclerosis biomarkers by proteomics

“…1A). A SELDI mass peak of this size was previously shown to be C-reactive protein (CRP) 16,17. We confirmed this mass spectrometry result using a commercial CRP ELISA and CSF from 41 ALS and 20 age-matched HC subjects used in the SELDI-TOF-MS analysis (Fig.…”

“…The 23,030 Da mass spectral peak exhibited the single best sensitivity and specificity for ALS and has been reported to be C-reactive protein (CRP), an acute-phase inflammatory protein 17,19. CRP was not detected as a potential biomarker in our prior study due to the mass range cut-off of 20 kDa used for that study.…”

Mass spectrometry of peptides and proteins from human blood

“…Mass spectrometry compatible, or acid-labile, detergents may also provide specificity (Ross et al, 2002;Nomura et al, 2004). Another means to avoid excessive background would be to use the affinity receptor immobilized at a high density on a low total surface area with concomitantly less area available for non-specific binding (Weinberger, Morris, & Pawlak, 2000;Weinberger et al, 2002;de Seny et al, 2008), such as a 96-well plate used in ELISA assays (Kulasingam et al, 2008), PVDF MASS SPECTROMETRY OF BLOOD & membrane (Chen et al, 2007b) et al, 2008). Antigen-antibody complexes in the 150-400 kDa range were chemically cross-linked and directly detected by MALDI mass spectrometry (Nazabal, Wenzel, & Zenobi, 2006).…”

Serum Amyloid A is a Novel Prognostic Biomarker in Hepatocellular Carcinoma

“…In addition, a number of previous studies proposed a direct correlation between SAA concentrations and tumor stage that a higher percent of raised SAA levels was found in patients with more advanced disease (Weinstein et al, 1984;Biran et al, 1986;Chen et al, 2007;Liu et al, 2007). And other authors have suggested SAA work as an useful indicator for signs of distant metastases in patients with RCC and LC (Ramankulov et al, 2008;Sung et al, 2011).…”