Improving Precursor Selectivity in Data-Independent Acquisition Using Overlapping Windows

Abstract

A major goal of proteomics research is the accurate and sensitive identification and quantification of a broad range of proteins within a sample. Data-independent acquisition (DIA) approaches that acquire MS/MS spectra independently of precursor information have been developed to overcome the reproducibility challenges of data-dependent acquisition and the limited breadth of targeted proteomics strategies. Typical DIA implementations use wide MS/MS isolation windows to acquire comprehensive fragment ion data. However, wide isolation windows produce highly chimeric spectra, limiting the achievable sensitivity and accuracy of quantification and identification. Here, we present a DIA strategy in which spectra are collected with overlapping (rather than adjacent or random) windows and then computationally demultiplexed. This approach improves precursor selectivity by nearly a factor of 2, without incurring any loss in mass range, mass resolution, chromatographic resolution, scan speed, or other key acquisition parameters. We demonstrate a 64% improvement in sensitivity and a 17% improvement in peptides detected in a 6-protein bovine mix spiked into a yeast background. To confirm the method’s applicability to a realistic biological experiment, we also analyze the regulation of the proteasome in yeast grown in rapamycin and show that DIA experiments with overlapping windows can help elucidate its adaptation toward the degradation of oxidatively damaged proteins. Our integrated computational and experimental DIA strategy is compatible with any DIA-capable instrument. The computational demultiplexing algorithm required to analyze the data has been made available as part of the open-source proteomics software tools Skyline and msconvert (Proteowizard), making it easy to apply as part of standard proteomics workflows. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13361-018-2122-8) contains supplementary material, which is available to authorized users.

PulseDIA: in-depth data independent acquisition mass spectrometry using enhanced gas phase fractionation

“…The PulseDIA is also different from conventional DIA method and DIA with gas-phase fractionation using consecutively separated windows 15,28 coupled with targeted data analysis by acknowledging the fact that peptide precursor ions are not evenly distributed long the m/z space. The PulseDIA utilizes discontinuous windows which allocate peptide precursor ions with different m/z evenly into each pulse injection.…”

“…In addition to the gas-phase separation, overlapping windows were designed for a DIA strategy which improves precursor selectivity without any loss in other key acquisition parameters. 15 Here, we present an alternative gas-phase separation PulseDIA-MS method, in which replicate sample injections are analyzed using differentially segmented DIA-MS methods in order to decrease window width and improve specificity and sensitivity. We demonstrated its applicability in identifying dysregulated proteins from cholangiocarcinoma (CCA), a rare malignant tumor composed of cells that resemble those of the biliary tract.…”

Partially overlapping sequential window acquisition of all theoretical mass spectra: A methodology to improve the spectra quality of veterinary drugs present at low concentrations in highly complex biological matrices

“…Waters has commercialized a scanning quadrupole DIA technique called “SONAR” . Fixed, but overlapping windows were employed, where the second SWATH window sequence was shifted around the m/z axis to produce an overlap with the window acquired during the first sequence …”

“…To the best of our knowledge, no peer‐reviewed papers have discussed the advantages and limitations of these new methodologies. The concept of a partial overlapping SWATH, as proposed in this paper, is an extension of the overlapping windows methodology . Partial overlapping SWATH not only switches between the original and another m/z ‐shifted SWATH window sequence, but employs a total of six m/z ‐shifted SWATH sequences.…”

“…12,13 Fixed, but overlapping windows were employed, where the second SWATH window sequence was shifted around the m/z axis to produce an overlap with the window acquired during the first sequence. 14 To the best of our knowledge, no peer-reviewed papers have discussed the advantages and limitations of these new methodologies. The concept of a partial overlapping SWATH, as proposed in this paper, is an extension of the overlapping windows methodology.…”