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An improved nanoflow RPLC-CZE-MS/MS system with high peak capacity and sensitivity for nanogram bottom-up proteomics

ABSTRACT: Novel mass spectrometry (MS)-based proteomic tools with extremely high sensitivity and high peak capacity are required for comprehensive characterization of protein molecules in mass-limited proteome samples. We previously reported a nanoflow RPLC-CZE-MS/MS (nanoRPLC-CZE-MS/MS) system for deep bottom-up proteomics of low micrograms of human cell samples (Yang et al. Anal. Chem. 2018, 90, 10479-10486). In this work, we further improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically via employing bovine serum albumin (BSA) treated sample vials, improving the nanoRPLC fraction collection procedure, and using a shorter capillary for fast CZE separation. The improved nanoRPLC-CZE produced a high peak capacity of 8500 for peptide separation. The improved system identified 6500 proteins from a MCF7 proteome digest starting with only 500-ng peptides using a Q-Exactive HF mass spectrometer. The system actually consumed roughly 100-ng peptides. The improved system produced a comparable number of protein identifications (IDs) to our previous system with 10-fold less sample consumption. The improved system is also comparable to the two-dimensional (2D) nanoRPLC-MS/MS system developed by the Mann’s group regarding the number of protein IDs starting with 500-ng human cell proteome digests but with 4-fold lower sample consumption. We further coupled single spot solid phase sample preparation (SP3) method to the improved nanoRPLC-CZE-MS/MS for bottom-up proteomics of 5000 HEK293T cells, resulting in 3689 protein IDs with the consumption of a peptide amount that corresponded to only roughly 1000 cells. This work represents the first study of a small number of human cells using CZE-MS/MS.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Breast Cancer

SUBMITTER: Liangliang Sun

LAB HEAD: Liangliang Sun

PROVIDER: PXD014392 | Pride | 2019-10-23

REPOSITORIES: Pride

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Improved Nanoflow RPLC-CZE-MS/MS System with High Peak Capacity and Sensitivity for Nanogram Bottom-up Proteomics.

Yang Zhichang Z Shen Xiaojing X Chen Daoyang D Sun Liangliang L

Journal of proteome research 20191022 11

Novel mass spectrometry (MS)-based proteomic tools with extremely high sensitivity and high peak capacity are required for comprehensive characterization of protein molecules in mass-limited samples. We reported a nanoRPLC-CZE-MS/MS system for deep bottom-up proteomics of low micrograms of human cell samples in previous work. In this work, we improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically via employing bovine serum albumin (BSA)-treated sample vials, improving the nanoRPLC ...[more]

PMID: 31610113

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Project description:Two-dimensional (2D) liquid chromatography (LC)-tandem mass spectrometry (MS/MS) are typically employed for deep bottom-up proteomics, and the state-of-the-art 2D-LC-MS/MS has approached over 8,000 protein identifications (IDs) from mammalian cell lines or tissues in 1-3 days of mass spectrometer time. Capillary zone electrophoresis (CZE)-MS/MS has been suggested as an alternative to LC-MS/MS for bottom-up proteomics. CZE-MS/MS and LC-MS/MS are complementary in protein/peptide ID from complex proteome digests because CZE and LC are orthogonal for peptide separation. In addition, the migration time of peptides from CZE-MS can be predicted accurately, which is invaluable for evaluating the confidence of peptide ID from the database search and even guiding the database search. However, the number of protein IDs from complex proteomes using CZE-MS/MS is still much lower than the state of the art using 2D-LC-MS/MS. In this work, for the first time, we established a strong cation exchange (SCX)-reversed phase LC (RPLC)-CZE-MS/MS platform for deep bottom-up proteomics. The platform identified around 8,200 protein groups and 65,000 unique peptides from a mouse brain proteome digest in 70 hours. The data represents the largest bottom-up proteomics dataset using CZE-MS/MS and provides a valuable resource for further improving the tool for prediction of peptide migration time in CZE. The peak capacity of the orthogonal SCX-RPLC-CZE platform was estimated to be around 7,000. SCX-RPLC-CZE-MS/MS produced comparable numbers of protein and peptide IDs with 2D-LC-MS/MS (8,200 vs. 8,900 protein groups, 65,000 vs. 70,000 unique peptides) from the mouse brain proteome digest using comparable instrument time. This is the first time that CZE-MS/MS showed its capability to approach comparable performance to the state-of-the-art 2D-LC-MS/MS for deep proteomic sequencing. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein and peptide IDs and combining those two methods improved the number of protein group and unique peptide IDs by nearly 10% and over 40%, respectively, compared with 2D-LC-MS/MS alone.

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