Maximizing LC-MS sensitivity and throughput in single-cell proteomics

Introduction

Proteomics has recently shifted towards profiling smaller sample quantities, including single-cell analysis. Although state-of-the-art liquid chromatography-mass spectrometry (LC-MS) technology has demonstrated unprecedented performance in investigating proteome changes, it lacks sensitivity for low-abundance peptides in single-cell proteomics (SCP) applications. Naturally, LC-MS sensitivity can be enhanced by utilizing ultralow LC flow rates (<20 nL/min) with small inner diameter columns (20-30 µm) and emitters, but this comes at the cost of decreased sample throughput and restricting the possibility to analyze large sample cohorts. Therefore, a balance between sensitivity and sample throughput is the prerequisite for large-scale single-cell sample profiling. To address this dilemma, we successfully developed a low-nanoflow UHPLC separation setup with a wide-window data-independent acquisition (WW-DIA) strategy to achieve deep proteome profiling in a high-throughput fashion.

Methods

We established a novel low nano flow LC-MS configuration running gradients at 100 nL/min on a Vanquish Neo UHPLC system with an Acclaim PepMap 100 C18 50 µm ID x 15 cm column (2 um dp) in direct injection and trap-and-elute workflows. Electrospray ionization was carried out via a 10 µm ID glass emitter into a FAIMS Pro interface operated at a single compensation voltage to reduce background ion interference, followed by data acquisition in an Orbitrap Exploris 480 or an Orbitrap Astral mass spectrometer.

Preliminary results

Seven chromatography methods were developed to permit fast sample loading, efficient column washing, and equilibration while maintaining a stable 100 nL/min flow during the gradient. This permits the analysis of up to 120 samples/day with a 8-min peptide elution window (i.e., 67% MS utilization), whilst also offering extremely high levels of sensitivity for routine SCP analysis. Using this optimized front-end configuration, we identified ~2,200 protein groups from 250 pg of standard protein digest using the WW-DIA strategy in a library-free search without booster channels. A proof of principle study indicated that >1,700 protein groups were identified and quantified from single-cell samples in a label-free experiment with negligible carryover at 100 cells/day (CPD) throughput. Furthermore, employing the Orbitrap Astral mass spectrometer significantly improved performance by doubling the protein identifications and quantification.

In conclusion, we established a high-performance LC-MS configuration for SCP by balancing the sensitivity and throughput, thus enabling the investigation of cellular heterogeneity in clinically and biologically relevant cellular populations.