Calculation of partial isotope incorporation into peptides measured by mass spectrometry

In this study, we use the characteristics of the so-called 'half decimal place rule' (HDPR) in order to accurately calculate the partial13C incorporation in peptides from enzymatic digested proteins. Due to the clade-crossing universality of proteins within bacteria, any available high-resolution mass spectrometry generated dataset consisting of tryptically-digested peptides can be used as reference.We used a freely available peptide mass dataset from Mycobacterium tuberculosis consisting of 315,579 entries. From this the error of estimated versus known heavy stable isotope incorporation from an increasing number of randomly drawn peptide sub-samples (100 times each; no repetition) was calculated. To acquire an estimated incorporation error of less than 5 atom %, about 100 peptide masses were needed. Finally, for testing the general applicability of our method, peptide masses of tryptically digested proteins from Pseudomonas putida ML2 grown on labeled substrate of various known concentrations were used and13C isotopic incorporation was successfully predicted. An easy-to-use script [1] was further developed to guide users through the calculation procedure for their own data series.Our method is valuable for estimating13C incorporation into peptides/proteins accurately and with high sensitivity. Generally, our method holds promise for wider applications in qualitative and especially quantitative proteomics.An important aspect of microbial ecology is to link specific microorganisms to microbially-driven processes in the natural environment [2]. Indeed, these questions can be addressed with more accuracy by using isotopically labeled substrates to follow metabolic carbon flux within microbial cultures. Subsequently, the incorporation of the label into the biomass may be followed by analysis of either fatty acids [3], deoxyribonucleic acid (DNA) [4] or ribonucleic acid (RNA) [5]. Unfortunately, those techniques are limited in their ability to resolve low levels of isoto