Dataset Information

Multicolumn Nanoflow Liquid Chromatography System with Accelerated Offline Gradient Generation for Robust and Sensitive Single-Cell Proteome Profiling

ABSTRACT: Peptide separations that combine high sensitivity, robustness, peak capacity and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with accelerated offline gradient generation. One binary pump generates gradients in an accelerated manner to support multiple analytical columns, and a single trap column interfaces with all analytical columns to reduce required maintenance and simplify troubleshooting. A high degree of parallelization is possible, as one sample undergoes separation while the next sample plus its corresponding mobile phase gradient are transferred into the storage loop and a third sample is loaded into a sample loop. Selective offline elution from the trap column into the sample loop prevents salts and hydrophobic species from entering the analytical column, thus greatly enhancing column lifetime and system robustness. With this design, samples can be analyzed as fast as every 20 min at a flow rate of just 40 nL/min with close to 100% MS utilization time, and continuously for as long as several months without column replacement. We utilized the system to analyze the proteomes of single cells from a multiple myeloma cell line upon treatment with the immunomodulatory imide drug lenalidomide.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Ryan Kelly

LAB HEAD: Ryan T. Kelly

PROVIDER: PXD049248 | Pride | 2025-05-06

REPOSITORIES: Pride

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Multicolumn Nanoflow Liquid Chromatography with Accelerated Offline Gradient Generation for Robust and Sensitive Single-Cell Proteome Profiling.

Xie Xiaofeng X Truong Thy T Huang Siqi S Johnston S Madisyn SM Hovanski Simon S Robinson Abigail A Webber Kei G I KGI Lin Hsien-Jung L HL Mun Dong-Gi DG Pandey Akhilesh A Kelly Ryan T RT

Analytical chemistry 20240625 26

Peptide separations that combine high sensitivity, robustness, peak capacity, and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with offline gradient generation. One binary pump generates gradients in an accelerated fashion to support multiple analytical columns, and a single trap column interfaces with all analytical columns to reduce required maintenance and simplify troubleshoot ...[more]

PMID: 38915247

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Project description:The sensitivity of single-cell proteomics (SCP) has increased dramatically in recent years due to advances in experimental design, sample preparation, separations and mass spectrometry instrumentation. However, further increasing the sensitivity of SCP methods and instrumentation will enable the study of proteins within single cells that are expressed at copy numbers too small to be measured by current methods. Here we further increase SCP sensitivity by combining efficient nanoPOTS sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to extend the achievable proteome coverage for single cells to >3,000 for single-cell data analyses. WWA is based on data-dependent acquisition (DDA) but employs larger precursor isolation windows to intentionally co-isolate and co-fragment additional precursors along with the selected precursor. The resulting chimeric MS2 spectra are then resolved using the CHIMERYS search engine within Proteome Discoverer 3.0. Compared to standard DDA workflows, WWA employing isolation windows of 8-12 Th increases peptide and proteome coverage by ~28% and ~39%, respectively. For a 40 min LC gradient operated at ~15 nL/min, we identified an average of 2,150 proteins per single-cell-sized aliquots of protein digest directly from MS2 spectra, which increased to an average 3,524 proteins including proteins identified with MS1-level feature matching. Reducing the active gradient to 20 min resulted in only a 10% decrease in proteome coverage. We also compared performance of WWA with DIA underperformed relative to WWA in terms of proteome coverage, especially with faster separations. Average proteome coverage for single HeLa and K562 cells was respectively 1,758 and 1,642 based on MS2 identifications with 1% false discovery rate and 3042 and 2891 with MS1 feature matching. As such, WWA combined with efficient sample preparation and rapid separations extends the depths of the proteome that can be studied at the single-cell level.

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	150_longitivity_0-4ng_test.msf	Msf
	2ng_2x_01.raw	Raw
	2ng_2x_02.raw	Raw
	2ng_4x_01.raw	Raw
	2ng_4x_02.raw	Raw