Introduction
Targeted analysis of DIA is a powerful mass spectrometric approach for comprehensive, reproducible and precise proteome quantitation. It provides valuable insight into biological processes or enables the discovery of novel biomarkers. Today, identification of the majority of the expressed proteins can now be achieved. With these achievements in the identification, the reproducibility and quantitative accuracy and precision have become increasingly important. Here, we optimize data-independent acquisition (DIA) on a Thermo Scientific™ Q Exactive™ HF mass spectrometer for quantification at the MS1 level. DIA data can be processed in a targeted mode based on MS2 fragment information. MS1 scans have potentially a higher sensitivity since the peptide precursor is not fragmented in multiple fragments. Novel methods using high resolution full range and segmented MS1 enable high precision quantification on MS1. Here we perform quantification of over 60,000 peptide precursors on MS1 level and benchmark MS1 based interference correction implemented in Spectronaut.
Methods
Protein samples for HeLa were prepared using the FASP protocol. Biognosys’ iRT kit was spiked into the samples before injection. The samples were acquired on a Q Exactive HF mass spectrometer. Shotgun runs were performed and searched using MaxQuant software. Spectral libraries were generated with Biognosys’ Spectronaut. DIA methods were acquired with varying gradients lengths. Targeted analysis of DIA runs was performed using Spectronaut.
Novel Aspects
Single shot DIA method using high resolution MS1 scan information for quantification.