Quantitative Profiling of Proteins in Complex Mixtures Using Liquid Chromatography and Mass Spectrometry

TLDR

In theory, peptide peak areas should correlate with their concentrations, so the peak areas of peptides from a single protein should reflect that protein’s concentration. The study aims to determine whether LC/MS data of tryptic digests can quantify proteins, testing this by analyzing varying amounts of myoglobin and proposing a combined identification‑profiling method. LC/MS was used to analyze tryptic digests of myoglobin across a 10 fmol–100 pmol range and to spike horse myoglobin into human serum for evaluation. Peak areas correlate linearly with protein concentration (r² = 0.991), the method works in complex mixtures like human serum with ratio errors ≤ 16%, and it offers a broadly applicable way to compare global protein expression. Keywords: mass spectrometry, protein quantification, protein identification.

Abstract

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples. Keywords: mass spectrometry • protein quantification • protein identification