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Deep single-shot nanoLCMS proteome profiling with 1500 bar UHPLC system, long fully porous columns, and HRAM MS

ABSTRACT: This study demonstrates how the latest UHPLC (ultra-high-performance liquid chromatography) technology can be combined with high-resolution accurate-mass (HRAM) mass spectrometry (MS) and long columns packed with fully porous particles to improve bottom-up proteomics analysis with nano-flow liquid chromatography mass-spectrometry (nanoLCMS) methods. The increased back pressures from the UHPLC system enabled the use of 75 µm I.D. x 75 cm columns packed with 2 µm particles at a typical 300 nL/min flow rate as well as elevated and reduced flow rates. The constant pressure pump operation at 1500 bar reduced sample loading and column washing/equilibration stages and overall overhead time that maximizes MS utilization time. The versatility of flow rate optimization to balance the sensitivity, throughput with sample loading amount, and capability of using longer gradients contribute to a greater number of peptide and protein identifications for single-shot bottom-up proteomics experiments. The routine proteome profiling and precise quantification of >7,000 proteins with single-shot nanoLC-MS analysis open possibilities for large-scale discovery studies with deep dive into the protein level alterations.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Brain Glioblastoma Multiforme,Cervix Carcinoma

SUBMITTER: Alexander Boychenko

LAB HEAD: Runsheng Zheng

PROVIDER: PXD035665 | Pride | 2022-09-12

REPOSITORIES: Pride

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Project description:In the light of the ongoing single-cell revolution, scientific disciplines are combining forces with the ultimate goal to retrieve as much relevant data as possible from trace amounts of biological material. For single cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, LC separation, MS/MS data acquisition and data analysis. To demonstrate the potential for single cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar array columns with state-of-the-art Orbitrap MS/MS detection and FAIMS. As compared to the currently commercially available pillar array columns, this dedicated limited sample column has a reduced cross section (factor of 2) and micro pillar dimensions that have been further downscaled (also by a factor of 2; 2.5 µm instead of 5 µm diameter), resulting in improved chromatography at reduced void times. A dilution series ranging from 5 to 0.05 ng/µL was prepared using trace amounts of PEG in the sample solvent to reduce adsorptive effects in the autosampler vial, sample stability up to 24h after dilution was demonstrated. Comparative processing of the MS/MS data with different database search algorithms (with and without second search feature activated and with and without rescoring based on fragmentation pattern prediction) pointed out the benefits of using Sequest HT together with INFERYS when analyzing samples at a concentration below 1 ng/µL. On average (data from quadruplicate runs), we were able to successfully identify 2855 unique protein groups from just 1 ng of HeLa lysate and this using a 60 min non-linear LC solvent gradient at a flow rate of 250 nL/min, hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10 (2436 proteins identified from 10 ng of HeLa lysate in 2019). By successfully identifying 1486 and 2210 proteins (average values, quadruplicates) from as little as 250 and 500 pg of HeLa lysate respectively, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.

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Deep single-shot nanoLCMS proteome profiling with 1500 bar UHPLC system, long fully porous columns, and HRAM MS

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