Accurate proteome-wide protein quantification from high-resolution 15N mass spectra

Experimental methods for proteome-wide quantification using liquid chromatography coupled mass spectrometry (LC-MS) rest heavily on computational methods that analyze mass spectra for peptide and protein quantification [1]. Computational analysis typically relies on two main components: (1) a peptide identification algorithm that assigns amino acid sequences to fragmentation spectra by searching a database of theoretical peptides obtained by an in silico digest of an organism's proteome; and (2) a quantification algorithm that collects peaks across mass spectra in the form of extracted ion chromatograms (XICs) over the duration of chromatographic elution of peptides.These algorithms have been shown to produce robust and accurate peptide quantification results across a wide range of quantification strategies. They are an important part of the data analysis employed in a widely used experimental approach called stable isotope labeling by amino acids in cell culture (SILAC) [2,3]. In most typical applications of this approach, a sample in which stable isotope-labeled amino acids are incorporated metabolically by an organism is compared to an unlabeled sample from a different experimental condition. Because both samples are combined prior to protein extraction, the samples are subjected to the same extraction, sample handling, digestion, chromatography, and ionization conditions. This eliminates much of the technical variation between individual samples. Consequently, the final relative abundance measurements, in the form of ratios of the areas of paired XICs from both the unlabeled and labeled peptides, are highly accurate. To ensure that only labeled amino acids are incorporated into proteins, amino acid auxotrophs are typically employed in conjunction with labeled amino acids, rendering this strategy unavailable for prototrophic microorganisms, such as wild-type bacteria and yeast.15N-labeling through the metabolic incorporation of a single source of labeled nitrogen