Combining Precursor and Fragment Information for Improved Detection of Differential Abundance in Data Independent Acquisition

Abstract

In bottom-up, label-free discovery proteomics, biological samples are acquired in a data-dependent (DDA) or data-independent (DIA) manner, with peptide signals recorded in an intact (MS1) and fragmented (MS2) form. While DDA has only the MS1 space for quantification, DIA contains both MS1 and MS2 at high quantitative quality. DIA profiles of complex biological matrices such as tissues or cells can contain quantitative interferences, and the interferences at the MS1 and the MS2 signals are often independent. When comparing biological conditions, the interferences can compromise the detection of differential peptide or protein abundance and lead to false positive or false negative conclusions.We hypothesized that the combined use of MS1 and MS2 quantitative signals could improve our ability to detect differentially abundant proteins. Therefore, we developed a statistical procedure incorporating both MS1 and MS2 quantitative information of DIA. We benchmarked the performance of the MS1-MS2-combined method to the individual use of MS1 or MS2 in DIA using four previously published controlled mixtures, as well as in two previously unpublished controlled mixtures. In the majority of the comparisons, the combined method outperformed the individual use of MS1 or MS2. This was particularly true for comparisons with low fold changes, few replicates, and situations where MS1 and MS2 were of similar quality. When applied to a previously unpublished investigation of lung cancer, the MS1-MS2-combined method increased the coverage of known activated pathways.Since recent technological developments continue to increase the quality of MS1 signals (e.g. using the BoxCar scan mode for Orbitrap instruments), the combination of the MS1 and MS2 information has a high potential for future statistical analysis of DIA data.

Emerging mass spectrometry-based proteomics methodologies for novel biomedical applications

“…With this methodology, Meier et al [57] detected more than 10 000 proteins in only 100 min with sensitivity into the low-attomolar range. While BoxCar approaches were originally developed to augment DDA scan types, the principle can also be applied to DIA [58].…”

Analyzing Ribosome Remodeling in Health and Disease

“…[118,119] DIA analysis can also benefit from incorporating MS2-level data in order to improve the accuracy of label-free quantification. [120] With isobaric labeling, peptides of each sample are covalently labeled with a sample-specific mass tag. Mass tags are comprised of a reporter ion, a balance group and a reactive group, which is usually an amine reactive group (NHS group) that binds to primary amines from the peptides.…”

Phosphoproteomics Meets Chemical Genetics: Approaches for Global Mapping and Deciphering the Phosphoproteome

“…This allows each phosphopeptide to be quantified from its MS1 precursor ion signal. It should be noted that, although not every phosphopeptide may have been subjected to MS2 fragmentation in each sample, it still remains quantifiable [ 152 , 154 , 155 , 156 ].…”