Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry

Abstract

Data independent acquisition (DIA) mass spectrometry is a powerful technique that is improving the reproducibility and throughput of proteomics studies. Here, we introduce an experimental workflow that uses this technique to construct chromatogram libraries that capture fragment ion chromatographic peak shape and retention time for every detectable peptide in a proteomics experiment. These coordinates calibrate protein databases or spectrum libraries to a specific mass spectrometer and chromatography setup, facilitating DIA-only pipelines and the reuse of global resource libraries. We also present EncyclopeDIA, a software tool for generating and searching chromatogram libraries, and demonstrate the performance of our workflow by quantifying proteins in human and yeast cells. We find that by exploiting calibrated retention time and fragmentation specificity in chromatogram libraries, EncyclopeDIA can detect 20–25% more peptides from DIA experiments than with data dependent acquisition-based spectrum libraries alone.

Removing the Hidden Data Dependency of DIA with Predicted Spectral Libraries

“…This accurate prior filtering makes the statistical burden of false targets in the wide window DIA surmountable again. Notably, due to instrument limitations this “Precursor Acquisition Independent From Ion Count” (PAcIFIC) can currently only be performed by means of gas phase fractionation (GPF), i.e., sampling different m/z regions separately . Still, the added acquisition depth and specificity allows for 88k (DDA), 47k (FASTA), and 95k (predicted) doubly and triply charged peptide detections as reported by the software, corresponding to 84k, 44k, and 90k peptidoforms in six narrow window GPF DIA runs of a HeLA cell lysate (Figure S4, Supporting Information).…”

“…Additionally, the peptide detections in narrow window DIA can be translated into novel and integrated PQPs, which are calibrated to the specific LC–MS system and are specific to DIA (Figure 1A). This approach was recently made readily applicable as chromatogram libraries: DIA libraries of narrow window DIA peptide detections comprising their calibrated PQPs . Such chromatogram libraries outperform direct wide window DIA extraction for every source library.…”

“…Here, we compare the effect of using libraries from different origins on peptide‐centric approaches, by assessing their qualitative and quantitative performance on a public wide window (10–20 m/z ) DIA dataset of HeLA cells ( Figure ). Three basic spectral libraries were used here, with PQPs derived from a) an experimental DDA dataset, b) a protein sequence database (FASTA), and c) a predicted spectral dataset.…”

“…Each of these three libraries can be used directly as a source library, or can be converted into a DIA library by using them first on a narrow window (2 m/ z) DIA dataset of the sample. The resulting six possible libraries can all be used alike by the EncyclopeDIA software to identify and quantify wide window DIA data . We define in the further text (i) peptide detections as being reported by the software above 1% FDR, (ii) peptidoforms as having deconvoluting charge states and (iii) robust peptides as being detected in three separate runs with at least three transitions.…”

R2‐P2 rapid‐robotic phosphoproteomics enables multidimensional cell signaling studies

“…The MS measurements resulted in 82 injections with 60‐min peptide separation gradients; together with the sample preparation (starting from cell pellets), the experiment was completed in 8 days (Fig A). For the analysis of the acquired data, we used Skyline to generate the spectral library from the DDA runs (MacLean et al , ), EncylopeDIA to generate the chromatogram library from the narrow‐window DIA runs (Searle et al , ), and Thesaurus to identify, site‐localize, and quantify phosphopeptides in the wide‐window DIA runs (Searle et al , ).…”