Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry

Abstract

Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed nanoPOTS platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest generation mass spectrometer. Following nanoPOTS sample preparation, protein digests from single cells were separated using a 20 μm i.d. in-housepacked nanoLC column. Separated peptides were ionized using an etched fused-silica emitter capable of stable operation at the ∼20 nL/min flow rate provided by the LC separation. Ultrasensitive LC−MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by tandem mass spectrometry (MS/MS) from single HeLa cells, and 874 protein groups were identified using the Match Between Runs feature of MaxQuant. This represents an >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger bore (30 μm i.d.) LC columns coupled to a previous-generation Orbitrap Fusion Lumos mass spectrometer.

Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell

“…When coupled with uorescence activate cell sorting and a custom autosampler, nanoPOTS has been used to analyse >150 single cells with excellent run-to-run reproducibility. 9 Ultrasensitive separations have been realized by reducing total ow rates to the low-nanolitre-per-minute range using capillary electrophoresis 10,11 or narrow-bore liquid chromatography (LC) with either open tubular 12,13 or packed columns, 14,15 providing reduced solvent contamination and improved ionization efficiency at the electrospray source.…”

Single-cell Proteomics: Progress and Prospects

“…We have since further miniaturized our LC separations for single-cell proteomics to 20-µm-i.d. columns operating at ~20 nL/min (Figure 2A), and the average MS/MS-derived identifications increased by >40% to nearly 300 protein groups using the same cell type, preparation method and mass spectrometer (27). This (50).…”

“…In analyzing 2 ng aliquots of bacterial lysate tryptic digest, we identified nearly 3 times more unique peptides using an Orbitrap Fusion Lumos compared to an LTQ Orbitrap XL mass spectrometer (49). More recently, we observed an increase in peptide and protein coverage of 36% and 20%, respectively, when analyzing single HeLa cells on the latest-generation Orbitrap Eclipse mass spectrometer compared to the Orbitrap Fusion Lumos (27). In all cases, MS acquisition settings tend to differ substantially from those used for bulk studies.…”

Optimizing accuracy and depth of protein quantification in experiments using isobaric carriers

“…To understand the major benefits of using the isobaric carrier, we began our analysis by considering the major challenges and bottlenecks for ultrasensitive MS analysis without using the isobaric carrier. Specifically, we aimed to evaluate the effects of the isobaric carrier on three stages of MS/MS data acquisition and analysis shown in We evaluated the efficiency of each of these three steps for 1-cell and 100-cell samples analyzed by Cong et al [18]. The data shown in Fig.…”

“…(1) detecting peptide-like features (precursor ions) during MS1 survey scans,(2) quantifying peptides from the small samples based on the reporter ions and (3) identifying peptide sequences from the fragment ions. Increasing input from 1 to 100 cells primarily benefits the identification rate of MS2 spectra Replicates of 1-cell and 100-cell HeLa samples were analyzed by label-free proteomics by Cong et al[18]. (a) The number of peptide-like features (unique isotopic envelopes resolved with respect to m/z and retention time with charge ≥ +2), the number of MS2 spectra acquired, and the number of peptide-spectral-matches (PSMs) determined by MaxQuant at 1% false-discovery rate (FDR).…”