Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform.

Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles. We explored the possibility of measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This work demonstrates the feasibility of high-throughput label-free single-cell proteomics.

Imaging and Direct Sampling Capabilities of Nanospray Desorption Electrospray Ionization with Absorption-Mode 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Nanospray desorption electrospray ionization mass spectrometry, a powerful ambient sampling and imaging technique, is herein coupled as an isolated source with 21 Tesla (21T) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Absorption-mode data, enabled by an external data acquisition system, is applied for improved mass resolution, accuracy, and dynamic range without compromising spectral acquisition rates. Isotopic fine structure (IFS) information is obtained from the ambient sampling of living Bacillus and Fusarium species, allowing for high confidence in molecular annotations with a resolution >830 k (at m/z 825). Tandem mass spectrometry experiments for biological samples are shown to retain the IFS in addition to gained fragmentation information, providing a further degree of annotation confidence from ambient analyses. Rat brain was imaged by nanospray desorption electrospray ionization (nano-DESI) 21T FTICR MS in ∼5 h using 768 ms transients, producing over 800 molecular annotations using the METASPACE platform and low-parts-per-billion mass accuracy at a spatial resolution of ∼25 × 180 µm. Finally, nano-DESI 21T FTICR MS imaging is demonstrated to reveal images corresponding to the IFS, as well as hundreds of additional molecular features (including demonstrated differences as low as 8.96 mDa) that are otherwise undetected by a more conventional imaging methodology.

Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography-Mass Spectrometry for High-Throughput Single-Cell Proteomics.

Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. We have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, we have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. We also developed a sample drying, extraction, and loading workflow to enable reproducible and reliable sample injection. The sequential analysis of 20 samples containing 10 ng tryptic peptides demonstrated high reproducibility with correlation coefficients of >0.995 between any two samples. The nanoPOTS autosampler can provide analysis throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, respectively. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an average of 256 proteins was identified from each cell and the number was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ∼77 single cells could be analyzed per day. We analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quantitative single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.

Ambient Metabolic Profiling and Imaging of Biological Samples with Ultrahigh Molecular Resolution Using Laser Ablation Electrospray Ionization 21 Tesla FTICR Mass Spectrometry.

Mass spectrometry (MS) is an indispensable analytical tool to capture the array of metabolites within complex biological systems. However, conventional MS-based metabolomic workflows require extensive sample processing and separation resulting in limited throughput and potential alteration of the native molecular states in these systems. Ambient ionization methods, capable of sampling directly from tissues, circumvent some of these issues but require high-performance MS to resolve the molecular complexity within these samples. Here, we demonstrate a unique combination of laser ablation electrospray ionization (LAESI) coupled with a 21 tesla Fourier transform ion cyclotron resonance (21T-FTICR) for direct MS analysis and imaging applications. This analytical platform provides isotopic fine structure information directly from biological tissues, enabling the rapid assignment of molecular formulas and delivering a higher degree of confidence for molecular identification.

Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses.

Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100 nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses. Graphical abstract A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.

Constant-Distance Mode Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Biological Samples with Complex Topography.

A new approach for constant-distance mode mass spectrometry imaging (MSI) of biological samples using nanospray desorption electrospray ionization (nano-DESI) was developed by integrating a shear-force probe with the nano-DESI probe. The technical concept and basic instrumental setup, as well as the general operation of the system are described. Mechanical dampening of resonant oscillations due to the presence of shear forces between the probe and the sample surface enabled the constant-distance imaging mode via a computer-controlled closed-feedback loop. The capability of simultaneous chemical and topographic imaging of complex biological samples is demonstrated using living Bacillus subtilis ATCC 49760 colonies on agar plates. The constant-distance mode nano-DESI MSI enabled imaging of many metabolites, including nonribosomal peptides (surfactin, plipastatin, and iturin) on the surface of living bacterial colonies, ranging in diameter from 10 to 13 mm, with height variations up to 0.8 mm above the agar plate. Co-registration of ion images to topographic images provided higher-contrast images. Based on this effort, constant-mode nano-DESI MSI proved to be ideally suited for imaging biological samples of complex topography in their native states.