Evaluation of diagonal-PASEF for data independent acquisition in proteomics

Introduction

Data independent acquisition became the most widely used method for proteomics due to its high level of sensitivity, reproducibility, and data completeness. To increase sensitivity and sample throughput, a lower number of broader isolation windows needs be applied, which leads to reduced selectivity. The combination with dispersive ion mobility separations like Trapped Ion Mobility (TIMS) prior to quadrupole isolation increases selectivity, however the quadrupole has to switch its isolation position in multiple steps to follow the mobility separation. A more efficient way to sample the mass-mobility range is to use a scanning quadrupole, that can continuously follow the ion cloud eluting from the TIMS device (diagonal-PASEF).

Methods

Diagonal-PASEF is enabled on recent instruments of the timsTOF series (Bruker, Bremen), running the latest instrument control software. Peptide identifications as well as quantitative reproducibility among technical replicates were evaluated with dilution series of human cell line digests (K562, Promega and HeLa, Pierce) in the pg to µg range, separated by nanoHPLC (250nl/min). Depending on gradient length, 5cm or 25cm columns (0.075mm ID, IonOpticks, Melbourne) were used. Data were processed with Spectronaut 19 (Biognosys, Schlieren).

Results

Scanning vs. switching quadrupole isolation schemes were designed to cover the same m/z - mobility range. A widely used window scheme consists of 8 isolation windows of 25 m/z width, that change their mass position twice during an ion mobility scan, covering almost all peptide ions in a range of 400-1000 m/z. With a scanning quad, 4 windows, also of 25 m/z width, can be used to cover a very similar set of precursor ions. This doubles acquisition speed as well as ion usage, as individual peptides are fragmented twice as often over the course of a chromatographic peak, so improvements were expected for low sample amounts and/or fast gradients. For 0.25ng of diluted cell digests, diagonal-PASEF outperformed the stepping window scheme in this technical comparison. Diagonal-PASEF also slightly increased the number of identified peptides and proteins from real single HeLa cells when comparing to the current optimal high sensitivity dia-PASEF method. For high sample amounts, identifications were similar between classic and diagonal-PASEF; for very short gradients a slight improvement in the percentage of identified precursors with a coefficient of variation (CV) below 10 % resp. 20% was observed.