An Attempt to Quantitative Analysis for Clinical Proteomics Using Stable Isotope-labeled Small Organic Molecules and MALDI-TOF-MS

Abstract

Quantitative analysis of proteins is essential to proteomics. By modifying specific amino acid residues with stable isotope-labeled or unlabeled small organic molecules followed by 2D electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), it is possible to quantitatively analyze the relative abundance of two tryptic peptides hence proteins based on the ratio of the relative peak intensities between the two peptides modified with isotope-labeled and unlabeled small organic molecules. Recently we have developed a protocol for quantitative analysis of such peptides by MALDI-TOF-MS using synthetic iodoacetanilide (IAA) and ¹³C₆-labeled iodoacetoanilide (¹³C₆-IAA) as well as N-β-naphthyliodoacetamide (NBN) and D₇-N-β-naphthyliodoacetamide (D₇-NBN) which specifically react with sulfhydryl groups in cysteine residues. Here we show that this technique can be applied to quantitative analysis of synthetic peptides and proteins extracted from human serum samples and separated on 2D electrophoresis. Two clusters of peaks representing IAA- / ¹³C₆-IAA- or NBN- / D₇-NBN-modified tryptic peptides were observed to be 6 or 7 Da apart due to the presence of six ¹³Cs or seven deuteriums, respectively. The mass difference of 6 or 7 Da facilitated identification of the modified peaks in the MALDI spectra. Both characterization and quantification of peptides and proteins are possible by the use of these stable isotope-labeled small organic molecules combined with MALDI-TOF-MS. We are trying to apply this technique to the clinical proteomics for exploration of the biomarkers of some specific disease.