Abstract
The data described here provide a systematic performance evaluation of popular data-dependent (DDA) and independent (DIA) mass spectrometric (MS) workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying 9 phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from non-enriched pull-down samples. The data are associated to the research article ‘Evaluation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites׳ (Bauer et al., 2014) [1]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.
Highlights
We found that all methods performed very in the absence of background proteins, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods
Different dilution series of peptides were analyzed using one dimensional liquid chromatography features separation and different data-dependent and independent mass spectrometry workflows to assess their limits of quantification
HeLa S3 were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) and penicillin–streptomycin (100 IU/ ml and 100 μg/ml, respectively, GIBCO) at 37 1C in a 5% CO2 atmosphere in a humidified incubator. 107 cells were collected by centrifugation and cell pellets washed twice with PBS
Summary
The general aim of this study was to assess the capabilities of recently established data-dependent (DDA) and independent (DIA) LC–MS strategies [2] in terms of sensitivity and linear relative quantification range for a variety of different peptides in the presence and absence of a complex analytical background (see Fig. 1). To assess analytical performance under a more realistic scenario, we prepared the same dilution series with a complex human digest spiked into each sample (referred to as complex samples). This allowed us to precisely determine the impact of the analytical background on limit of detection (LOD), quantification (LOQ) and identification (LOI) for each peptide and MS-approach applied in this study. We generated dilution profile correlations for each peptide and MS-method and applied an established algorithm [3,4] to determine linear quantification ranges as well as identification, quantification and detection limits
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