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Integrated MICROFASP method with CZE based fractionation technique and nanoRPLC-ESI-MS/MS for comprehensive proteomics analysis of submicrogram sample

ABSTRACT: We report a loss-less two dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for comprehensive proteomics analysis of submicrogram sample. Protein digest was injected into the linear polyacrylamide coated capillary followed by CZE separation. The schemes to collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials followed by nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimized sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5,231 protein groups from 500 ng of a HeLa protein digest within 11.5 hours (90 min CZE fractionation plus 10 hours LC-MS analysis). Finally, the developed platform was used to analysis the protein digest prepared by MICROFASP method with 1 µg cell lysate as starting material. 3796 (N=2, RSD=4.95%) protein groups and 20,577(N=2, RSD=7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 hours. The results indicated that combination of MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of submicrogram biological sample.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

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

DISEASE(S): Cervix Carcinoma

SUBMITTER: Zhenbin Zhang

LAB HEAD: Zhenbin Zhang

PROVIDER: PXD052735 | Pride | 2024-08-10

REPOSITORIES: Pride

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Integrated MICROFASP Method with CZE-Based Fractionation Technique and NanoRPLC-ESI-MS/MS for a Comprehensive Proteomics Analysis of a Submicrogram Sample.

Lu Gang G Ying Guojin G He Yu Y Li Yang Y Zhang Zhenbin Z

Journal of proteome research 20240716 8

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials ...[more]

PMID: 39013029

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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.

			Action	DRS
	350ng-4fractions-F-1.msf	Msf
	350ng-4fractions-F-1.raw	Raw
	350ng-4fractions-F-2.msf	Msf
	350ng-4fractions-F-2.raw	Raw
	350ng-4fractions-F-3.msf	Msf