High sensitivity limited material proteomics empowered by data-independent acquisition on linear ion traps

Abstract

In recent years, the concept of cell heterogeneity in biology has gained increasing attention, concomitant with a push towards technologies capable of resolving such biological complexity at the molecular level. While RNA-based approaches have long been the method of choice, advances in mass spectrometry (MS)-based technologies have led to the ability to resolve cellular proteomes within minute sample quantities and, very recently, even down to a single cell. Current limitations are the incomplete proteome depth achieved and low sample throughput, and continued efforts are needed to push the envelope on instrument sensitivity, improved data acquisition methods, and chromatography. For single-cell proteomics using Mass Spectrometry (scMS) and low-input proteomics experiments, the sensitivity of an orbitrap mass analyzer can sometimes be limiting. Therefore, low-input proteomics and scMS could benefit from linear ion traps, which provide faster scanning speeds and higher sensitivity than an orbitrap mass analyzer, however, at the cost of resolution. We optimized and improved an acquisition method that combines the orbitrap and linear ion trap, as implemented on a tribrid instrument, while taking advantage of the high-field asymmetric waveform ion mobility spectrometry (FAIMS) pro interface, with a prime focus on low-input applications. First, we compared the performance of orbitrap- versus linear ion trap mass analyzers. Subsequently, we optimized critical method parameters for low-input measurement by data-independent acquisition (DIA) on the linear ion trap mass analyzer. We conclude that linear ion traps mass analyzers combined with FAIMS and WhisperTM flow chromatography are well-tailored for low-input proteomics experiments. They can simultaneously increase the throughput and sensitivity of large-scale proteomics experiments where limited material is available, such as clinical samples, cellular sub-populations, and eventually, scMS.

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

“…We adopt a DIA approach that solely relies on precursor level quantification to further enhance sensitivity and use our findings to establish the WISH-DIA method. In tribrid instruments, the LIT can also be used to increase sensitivity while keeping isolation windows narrow 43 . We also applied the HRMS1 modification to LIT and showed that it significantly boosted proteome coverage for low-input samples (Figure S3).…”

Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics

“…42 Makarov et al estimated ∼20+ charges were required to generate a detectable signal in OT, 43 indicating its sensitivity is >10-fold lower than LIT. Indeed, a few of recent studies have demonstrated that LIT can greatly improve low-input proteomics based on data-independent acquisition (DIA), 44,45 parallel reaction monitoring (PRM), 46 and synchronous precursor selection (SPS) MS3. 47 In the SPS-MS3 study, 47 Liu and co-authors used 250 ng, TMT 6-plex labeled peptides to compare the proteome coverages and quantification performance between LIT-MS3 and OT-MS3.…”