Segmentation of Precursor Mass Range Using “Tiling” Approach Increases Peptide Identifications for MS¹-Based Label-Free Quantification

Abstract

Label-free quantification is a powerful tool for the measurement of protein abundances by mass spectrometric methods. To maximize quantifiable identifications, MS1-based methods must balance the collection of survey scans and fragmentation spectra while maintaining reproducible extracted ion chromatograms (XIC). Here we present a method which increases the depth of proteome coverage over replicate data-dependent experiments without the requirement of additional instrument time or sample pre-fractionation. Sampling depth is increased by restricting precursor selection to a fraction of the full MS1 mass range for each replicate; collectively, the m/z segments of all replicates encompass the full MS1 range. Although selection windows are narrowed, full MS1 spectra are obtained throughout the method, enabling the collection of full mass range MS1 chromatograms such that label-free quantitation can be performed for any peptide in any experiment. We term this approach “binning” or “tiling” depending on the type of m/z window utilized. By combining the data obtained from each segment, we find that this approach increases the number of quantifiable yeast peptides and proteins by 31% and 52%, respectively, when compared to normal data-dependent experiments performed in replicate.

DeMix-Q: Quantification-Centered Data Processing Workflow

“…This result may herald a new strategy in label-free shotgun proteomics, emphasizing the acquisition of higher-quality MS 1 spectra and deeper proteome coverage rather than larger number of redundant MS/MS spectra. One way of exploring this new strategy is by segmenting the mass range for precursor selection (48); another one is by applying the exclusion list aggregated from previous runs to subsequent runs.…”

Neutron-Encoded Protein Quantification by Peptide Carbamylation

“…Proteomic samples from either mouse liver or yeast cultures were performed as previously described [1, 4, 5]. Isotopically labeled urea ( 15 N 2 or 13 C 1 ) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA).…”

“…Peptides were carbamylated by incubating at pH 8 with a large excess of aqueous 8 M urea at 80°C for 4 hours and remaining urea was removed by desalting (Online Resource 1) [6]. Fractionation of the murine peptides was performed by offline high pH reverse-phase separation [4]. Peptide mixtures were separated using self-prepared capillary LC columns and analyzed upon elution using an Orbitrap Elite mass spectrometer with conditions described elsewhere (Thermo Scientific, San Jose, CA) [1].…”

Bioinformatic Approaches to Increase Proteome Coverage

“…This technique has been termed binning or tiling (20), depending on whether the full MS1 range is divided into continuous sequential segments or in several smaller segments distributed over the lower, middle, and upper mass regions. An alternative fractionation technique that can be used in the m/z dimension is gas-phase fractionation (GPF).…”