Maximizing peptide identification events in proteomic workflows using data-dependent acquisition (DDA)

Abstract

Current analytical strategies for collecting proteomic data using data-dependent acquisition (DDA) are limited by the low analytical reproducibility of the method. Proteomic discovery efforts that exploit the benefits of DDA, such as providing peptide sequence information, but that enable improved analytical reproducibility, represent an ideal scenario for maximizing measureable peptide identifications in "shotgun"-type proteomic studies. Therefore, we propose an analytical workflow combining DDA with retention time aligned extracted ion chromatogram (XIC) areas obtained from high mass accuracy MS1 data acquired in parallel. We applied this workflow to the analyses of sample matrixes prepared from mouse blood plasma and brain tissues and observed increases in peptide detection of up to 30.5% due to the comparison of peptide MS1 XIC areas following retention time alignment of co-identified peptides. Furthermore, we show that the approach is quantitative using peptide standards diluted into a complex matrix. These data revealed that peptide MS1 XIC areas provide linear response of over three orders of magnitude down to low femtomole (fmol) levels. These findings argue that augmenting "shotgun" proteomic workflows with retention time alignment of peptide identifications and comparative analyses of corresponding peptide MS1 XIC areas improve the analytical performance of global proteomic discovery methods using DDA.

Fig. 1.

Analytical workflow for differential analysis of peptide MS1 XIC data. A, Retention time alignment was performed iteratively for peptides co-identified across sample sets. Retention time of peptides identified in example sample set X were aligned with retention time for peptides co-identified in example sample set Y until an R² > 0.99 was achieved. B, Peptide MS1 XIC areas were then integrated for peptides identified and/or inferred across all sample sets. Pseudo-2D gel images of nLC-MS/MS data depicts peptides independently identified in example sample sets X and Y (arrows) and corresponding peptide MS1 XIC areas integrated (red circles) between both sample sets following retention time alignment of co-identified peptides.

Fig. 2.

Comparison of peptide identification events versus successful peptide MS1 XIC integration events across a representative set of technical replicate injections. Three-way Venn diagrams detailing overlapping peptide identification events (A) versus successfully integrated, retention time aligned peptide MS1 XIC areas (B) across a set of three technical replicate injections of tryptic digests derived from highly hemolyzed mouse blood plasma. These results revealed a 30.5% increase in the number of measurable peptide events co-identified across replicate injections.

Fig. 3.

Quantitative analysis of bovine peptide MS1 XIC areas in a dilution series of six equimolar bovine standard proteins in immunodepleted mouse blood plasma. Plots of peptide MS1 XIC areas versus bovine standard concentration across a six-point dilution series for four representative bovine standard peptides corresponding to alpha casein (A), beta-lactoglobulin (B), carbonic anhydrase (C), and glutamate dehydrogenase (D). Results revealed that bovine standard peptides diluted in immunodepleted mouse blood plasma are quantifiable to low fmol ranges. Mean R² = 0.9 ± 0.33 for all bovine peptides analyzed.

Fig. 4.

Peptide identification events versus successfully integrated peptide MS1 XIC areas following comparative proteomic analyses of olfactory bulb synaptic density proteins obtained from Homer2 knockout and wild-type mice. A, Two-way Venn diagrams detailing overlapping peptide identification events (left) versus successfully integrated, retention time aligned peptide MS1 XIC areas (right) across three technical replicate injections of tryptic digests derived from Homer2 knockout and wild-type olfactory bulb synaptic density tissues. These data resulted in a 27.1% increase in the number of measurable peptide events co-identified across replicate injections. B, Knockout and wild-type peptide MS1 XIC areas for a successfully integrated, representative Homer2 peptide plotted using Skyline software (24).

Fig. 5.

Peptide identification events versus successfully integrated peptide MS1 XIC areas in comparative proteomic analyses of hemolyzed blood plasma. Two-way Venn diagrams detailing overlapping peptide identification events (A) versus successfully integrated, retention time aligned peptide MS1 XIC areas (B) across three technical replicate injections of tryptic digests derived from high and low hemolyzed mouse blood plasma. These results revealed a 17.8% increase in the number of measurable peptide events co-identified across replicate injections.