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 7,100 proteins when including the 717 proteins that were identified on the basis of a single peptide sequence. 7,739 proteins were identified in mouse tissues using 2-or-more peptides/protein and 8,121 when including the 382 proteins that were identified on the basis of 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 1,412 proteins that were identified on the basis of 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)

SUBMITTER: Lukas Reiter  

PROVIDER: MSV000084874 | MassIVE | Thu Jan 30 16:33:00 GMT 2020

SECONDARY ACCESSION(S): PXD005573

REPOSITORIES: MassIVE