Optimization of Experimental Parameters in Data-Independent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results

Abstract

Comprehensive, reproducible and precise analysis of large sample cohorts is one of the key objectives of quantitative proteomics. Here, we present an implementation of data-independent acquisition using its parallel acquisition nature that surpasses the limitation of serial MS2 acquisition of data-dependent acquisition on a quadrupole ultra-high field Orbitrap mass spectrometer. In deep single shot data-independent acquisition, we identified and quantified 6,383 proteins in human cell lines using 2-or-more peptides/protein and over 7100 proteins when including the 717 proteins that were identified on the basis of a single peptide sequence. 7739 proteins were identified in mouse tissues using 2-or-more peptides/protein and 8121 when including the 382 proteins that were identified based on a single peptide sequence. Missing values for proteins were within 0.3 to 2.1% and median coefficients of variation of 4.7 to 6.2% among technical triplicates. In very complex mixtures, we could quantify 10,780 proteins and 12,192 proteins when including the 1412 proteins that were identified based on a single peptide sequence. Using this optimized DIA, we investigated large-protein networks before and after the critical period for whisker experience-induced synaptic strength in the murine somatosensory cortex 1-barrel field. This work shows that parallel mass spectrometry enables proteome profiling for discovery with high coverage, reproducibility, precision and scalability.

Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1

“…To analyse the DIA‐MS data, we used both spectrum‐centric (named directDIA in Spectronaut, a method extracting pseudo‐ spectrum directly from DIA data without the need of an assay library) and peptide‐centric (based on SWATH assay library which contains mass spectrometric assays for 10 000 human proteins, named panHuman hereafter) approaches. Both approaches achieved substantial sensitivity when both peptide‐ and protein‐ FDR were strictly controlled at 1% (Figure A,B) .…”

A streamlined mass spectrometry-based proteomics workflow for large scale FFPE tissue analysis

“…S2F). With the recent increased scan speed of Orbitrap analyzers, this platform has become very attractive for DIA strategies [31]. In DIA, the entire sample complexity is in principle captured by cycling of the quadrupole selection window over the entire m/z range in predefined segments, thereby recording MS2 information irrespective of precursor intensity.…”

“…In DIA, the entire sample complexity is in principle captured by cycling of the quadrupole selection window over the entire m/z range in predefined segments, thereby recording MS2 information irrespective of precursor intensity. While proteome coverage was previously a major bottleneck for DIA single-run workflows, more than 7,000 quantified protein groups have been reported from human cell lines in single 2 h measurements [32]. This prompted us to combine our streamlined tissue workflow with state-of-the-art DIA analysis based on the same 100 min LC gradient as in DDA analysis (Methods).…”

Evaluation of Urinary Proteome Library Generation Methods on Data‐Independent Acquisition MS Analysis and its Application in Normal Urinary Proteome Analysis

“…Furthermore, DIA has been proven to tend to have high reproducibility, high validity, and deep proteome coverage. [5] DIA methods have been widely used in various biofluids such as plasma, [6] urine, [7] follicular fluid, [8] and bronchoalveolar lavage fluid. [9] The DIA strategy divides the entire mass range into a series of sequential windows and acquires fragment ions on the basis of the acquisition of fragment ions for all precursors.…”

“…Compared with DDA, DIA/SWATH was able to acquire information on low‐abundance ions. Furthermore, DIA has been proven to tend to have high reproducibility, high validity, and deep proteome coverage . DIA methods have been widely used in various biofluids such as plasma, urine, follicular fluid, and bronchoalveolar lavage fluid .…”