Authors: Pang, Ronald¹; Johnson, Philip²; Chan, Charles³; Kong, Ebenezer³; Chan, Anthony³; Sung, Joseph¹; Poon, Terence⁴

Source: Clinical Proteomics, Volume 1, Numbers 3-4, September 2004 , pp. 259-270(12)

Publisher: Humana Press

Abstract:

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been recently used to identify disease markers by directly profiling and quantifying the peptide/proteins in biological samples under different physiological or experimental conditions. The information of reproducibility of such quantitative profiling method has not been available. It is important to evaluate and reduce error from technical variation. In this study, an unbiased signal acquisition strategy was used to evaluate the effects of three sample-matrix spotting methods and two matrix chemicals, α-cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid, on the reproducibility of the peptide/protein signal intensities. The sandwich spotting method using 0.1% nitrocellulose coating film and CHCA gave the best quantitative results for the standard peptides and proteins with mass<66.5 kDa. The normalized signal intensities of the standard peptides and proteins were directly proportional to their concentrations with intra-assay (within-day) coefficient of variations (CVs) ranging from 6.5% to 17%. When analyzing serum peptides <6000 m/z, the interassay (between-days) CVs of all the evaluated peptide peaks were <15%. These data indicate that with the right MS analysis conditions, MALDI-TOF MS appears to be a feasible tool for directly profiling and quantifying the peptide/ proteins in biological samples.

Keywords: Matrix-assisted laser desorption/ionization time-o; quantitative proteomics; nitrocellulose; reproducibility; variation

Document Type: Research article

DOI: http://dx.doi.org/10.1385/CP:1:3-4:259

Affiliations: 1: Department of Medicine and Therapeutics, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, 2: Cancer Research UK Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham, England, 3: Department of Clinical Oncology, the Sir Y. K. Pao Center for Cancer, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, the People's Republic of China, 4: Department of Medicine and Therapeutics, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, Email: tcwpoon@cuhk.edu.hk

Publication date: 2004-09-01

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