Open AccessQuantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Author(s) -
Julián Gutiérrez,
Jill A. Dorocke,
Michael D. Knierman,
Valentina Gelfanova,
Richard E. Higgs,
Nicholas Lewin Koh,
John E. Hale
Publication year - 2005
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/05386su02
Subject(s) - mass spectrometry , chemistry , analyte , time of flight mass spectrometry , chromatography , matrix assisted laser desorption/ionization , analytical chemistry (journal) , matrix (chemical analysis) , quantitative analysis (chemistry) , quantitative proteomics , biological system , ionization , proteomics , desorption , biochemistry , ion , organic chemistry , adsorption , biology , gene
A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a “seed crystal” protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.