title: Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.
abstract: A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Zhenbin
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Yan
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D002412:Cations / Q000737:chemistry; D019075:Electrophoresis, Capillary; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D012822:Silicon Dioxide / Q000737:chemistry*; D052616:Solid Phase Extraction; D013451:Sulfonic Acids / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D002412:Cations; D010455:Peptides; D020543:Proteome; D013451:Sulfonic Acids; D012822:Silicon Dioxide
references.citation: J Chromatogr A. 2012 Oct 26;1261:121-8
references.pmid: 22542442
references.citation: Nat Biotechnol. 1996 Nov;14(11):1579-83
references.pmid: 9634825
references.citation: Anal Chem. 2012 Feb 7;84(3):1617-22
references.pmid: 22182061
references.citation: Rapid Commun Mass Spectrom. 2011 Aug 15;25(15):2124-30
references.pmid: 21710592
references.citation: Electrophoresis. 2009 Jan;30(1):230-48
references.pmid: 19156661
references.citation: Electrophoresis. 2009 Jan;30(1):215-29
references.pmid: 19101931
references.citation: J Chromatogr A. 2008 Mar 14;1184(1-2):504-41
references.pmid: 18035364
references.citation: J Biochem Biophys Methods. 2007 Mar 10;70(2):289-97
references.pmid: 17196661
references.citation: J Chromatogr A. 2007 Jan 26;1140(1-2):205-12
references.pmid: 17174962
references.citation: Talanta. 2015 Jun 1;138:117-22
references.pmid: 25863379
references.citation: J Chromatogr A. 1997 Feb 28;763(1-2):295-306
references.pmid: 9129327
references.citation: Electrophoresis. 2011 Jan;32(1):105-15
references.pmid: 21171117
references.citation: Anal Chem. 2010 Nov 15;82(22):9476-83
references.pmid: 21028888
references.citation: Rapid Commun Mass Spectrom. 2010 Sep 15;24(17):2554-60
references.pmid: 20740530
references.citation: J Chromatogr A. 2009 Jun 19;1216(25):4933-40
references.pmid: 19446825
references.citation: Angew Chem Int Ed Engl. 2014 Dec 8;53(50):13931-3
references.pmid: 25346227
references.citation: Anal Chem. 2014 Jul 1;86(13):6331-6
references.pmid: 24852005
references.citation: Anal Chem. 2014 Apr 15;86(8):3817-24
references.pmid: 24666247
references.citation: J Chromatogr A. 2014 Mar 28;1335:43-60
references.pmid: 24210300
references.citation: Rapid Commun Mass Spectrom. 2014 Feb 15;28(3):305-10
references.pmid: 24375882
references.citation: Angew Chem Int Ed Engl. 2013 Dec 16;52(51):13661-4
references.pmid: 24173663
references.citation: J Pharm Biomed Anal. 2014 Jan;87:130-41
references.pmid: 23787353
references.citation: Anal Chem. 2013 Mar 5;85(5):2569-73
references.pmid: 23394296
references.citation: Proteomics. 2012 Oct;12(19-20):2991-3012
references.pmid: 22888086
references.citation: Anal Chem. 2012 Oct 16;84(20):8505-13
references.pmid: 23004022
references.citation: J Chromatogr A. 2012 Sep 21;1256:136-43
references.pmid: 22884132