Data-Independent Acquisition Shortens the Analytical Window of Single-Cell Proteomics to Fifteen Minutes in Capillary Electrophoresis Mass Spectrometry

Capillary zone electrophoresis (CE) combines high separation power, scalability, and speed to limited proteome analyses by mass spectrometry (MS). However, compressed separation in CE challenges the duty cycle of tandem MS, even during data-independent acquisition (DIA). To help remedy this limitation, we introduce the concept of electrophoresis-correlative (Eco) data acquisition for CE-MS. We recognize CE electrospray ionization (ESI) to sort peptide ions into reproducible mass-to-charge (m/z) vs migration time (MT) trends in the solution phase, before subsequent ionization and m/z analysis. We proposed that such a correlation can be leveraged to improve the economy of data acquisition. We test this hypothesis using DIA frames that are tailored to the observed m/z−MT trends. The resulting Eco-DIA method substantially improves the bandwidth utilization of tandem MS during CE-MS. In proof-of-principle studies, Eco-DIA identified and quantified ∼38% more proteins from 1 ng of the HeLa proteome digest compared to the classical DIA, without the assistance of a project-specific tandem MS spectral library. Eco-DIA was able to quantify ∼51% more proteins with <10% coefficient of variation vs the control DIA approach. Based on label-free quantification, the proteins that were exclusively measured by Eco-MS occupied the lower dynamic range of the detected proteome concentration, revealing sensitivity enhancement. In addition to marking the inception of Eco-MS, this work lays the foundation for the development of next-generation data acquisition strategies that leverage electrophoretic ion sorting for highsensitivity proteomics.

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Development and Validation of RoboCap, a Robotic Capillary Platform to Automate Capillary Electrophoresis Mass Spectrometry En Route to High-Throughput Single-Cell Proteomics

Jia,

Nemes

2024

Anal. Chem.

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Current developments in single-cell mass spectrometry (MS) aim to deepen proteome coverage while enhancing analytical speed to study entire cell populations, one cell at a time. Custom-built microanalytical capillary electrophoresis (μCE) played a critical role in the foundation of discovery single-cell MS proteomics. However, requirements for manual operation, substantial expertise, and low measurement throughput have so far hindered μCE-based single-cell studies on large numbers of cells. Here, we design and construct a robotic capillary (RoboCap) platform that grants single-cell CE-MS with automation for proteomes limited to less than ∼100 nL. RoboCap remotely controls precision actuators to translate the sample to the fused silica separation capillary, using vials in this work. The platform is hermetically enclosed and actively pressurized to inject ∼1−250 nL of the sample into a CE separation capillary, with errors below ∼5% relative standard deviation (RSD). The platform and supporting equipment were operated and monitored remotely on a custom-written Virtual Instrument (LabView). Detection performance was validated empirically on ∼5−250 nL portions of the HeLa proteome digest using a trapped ion mobility mass spectrometer (timsTOF PRO). RoboCap improved CE-ESI sample utilization to ∼20% from ∼3% on the manual μCE, the closest reference technology. Proof-of-principle experiments found proteome identification and quantification to robustly return ∼1,800 proteins (∼13% RSD) from ∼20 ng of the HeLa proteome digest on this earlier-generation detector. RoboCap automates CE-MS for limited sample amounts, paving the way to electrophoresis-based highthroughput single-cell proteomics.

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Data-Independent Acquisition Shortens the Analytical Window of Single-Cell Proteomics to Fifteen Minutes in Capillary Electrophoresis Mass Spectrometry

Cited by 4 publications

References 64 publications

Electrophoresis-Correlative Ion Mobility Deepens Single-cell Proteomics in Capillary Electrophoresis Mass Spectrometry

Electrophoresis-Correlative Ion Mobility Deepens Single-cell Proteomics in Capillary Electrophoresis Mass Spectrometry

Electrophoresis-Correlative Data-Independent Acquisition (Eco-DIA) Improves the Sensitivity of Mass Spectrometry for Limited Proteome Amounts

Development and Validation of RoboCap, a Robotic Capillary Platform to Automate Capillary Electrophoresis Mass Spectrometry En Route to High-Throughput Single-Cell Proteomics

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