Development of a high-throughput IMS MS approach for analyzing
CZE-nanoESI-SLIM-IMS-MS Platforms for Comprehensive ......Figure 2: A) Total ion traces for CZE-MS...
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Roza Wojcik, Ian K. Webb, Derek F. Hopkins , Spencer A. Prost, Randolph V. Norheim, Daniel Orton, Sandilya V. B. Garimella, Liulin Deng, Ahmed M. Hamid, Ryan Kelly, Yehia Ibrahim, Erin S. Baker, Richard D. Smith Introduction Overview Methods Results Acknowledgements Portions of this research were supported by grants from the National Institute of General Medical Sciences (P41 GM103493), National Institute of Environmental Health Sciences of the NIH (R01ES022190) and the Laboratory Directed Research and Development Program at Pacific Northwest National Laboratory. This research utilized capabilities developed by the Pan-omics program (funded by the U.S. Department of Energy Office of Biological and Environmental Research Genome Sciences Program) and by the National Institute of Allergy and Infectious Diseases under grant U19AI106772. This work was performed in the W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a DOE national scientific user facility at the Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the DOE under contract DE-AC05-76RL0 1830. References [1] Wojcik et al, RCMS. 2010; 24: 2554–2560 [2] Webb et al, Anal. Chem. 2014, 86, 9169−9176 [3] Hamid et al, Anal. Chem. 2015, 87, 11301−11308 [4] Garimella et al, Analyst, 2015, 140, 6845–6852 [5] TOD 4:10 pm: Smith et al; Ultrahigh IMS resolution using novel traveling wave approaches and long serpentine path length SLIM modules [6] ThOH 8:50 am Webb, et al; Analysis of Protein Structural Changes with High Resolution Structures for Lossless Ion Manipulations (SLIM) Ion Mobility-Mass Spectrometry [7] WOD 2:30 pm: Garimella et al; Fundamentals of Ion Dynamics in Structures for Lossless Ion Manipulations (SLIM) Conclusions Enhancing peak capacity for peptide separations using extended path length SLIM-TW-IMS-TOF-MS CONTACT: Roza Wojcik, Ph.D. Biological Sciences Division Pacific Northwest National Laboratory E-mail: [email protected] • Capillary zone electrophoresis (CZE) was coupled with ion mobility and mass spectrometry (IMS-MS) to evaluate advantages of the three dimensional measurements • CZE-nanoESI-IMS-MS was used to address proteomic challenges including: - Ultra-sensitive and efficient separations - Enhancement of peak capacity - Separation & structural characterization of intact proteins in denatured and native conditions Progress towards comprehensive proteome characterization relies on the development of high resolution front end separation techniques, efficient ionization and improved ion transmission mass spectrometric analysis. In this work we couple CZE-nanoESI with various IMS platforms, including: • a 1-m drift tube-IMS-MS • a high charge capacity Structures for Lossless Ion Manipulation (SLIM-IMS) travelling wave (TW) device • a high resolution SLIM-IMS TW device with a variable path length. CZE coupled with the nanoESI sheath flow interface (see Figure 1A) allows for sensitive separations of peptides and intact proteins in various background electrolyte conditions. Coupling CZE-nanoESI with IMS-MS provides opportunities to address the challenges of proteomic analyses, including enhancement of peak capacity and sensitivity, analysis of isomers, and solution phase/gas phase structural conformations. www.omics.pnl.gov Career Opportunities: For openings in the Integrative Omics Group at PNNL please visit http:// omics.pnl.gov/careers CZE-nanoESI-SLIM-IMS-MS Platforms for Comprehensive, Ultrasensitive Proteome Analyses • CZE separations were performed on an in-house constructed CE system. • The CE-MS interface [1] employed a 15 μm i.d. tapered borosilicate glass emitter and an electrokinetically pumped sheath liquid with 300 μm between the etched capillary exit and the ESI emitter • Experimental conditions used were as follows: - CZE-ESI of 1 μM phosphopeptide isomers (Figure 2): 30-cm long, 30 μm i.d., capillary with 200 mM formic acid as background electrolyte (BGE); 10 mM formic acid, 20% methanol as sheath liquid (SL); 19 kV separation, - CZE-ESI for 10 μM BSA digest (Figure 3): 28-cm long, 20 μm i.d. capillary with 100 mM acetic acid as BGE and 20 mM acetic acid, 20% methanol as SL; 28 kV separation - CZE-ESI of intact proteins in acidic condition (Figure 4-A-C): 45 cm long 30 μm i.d., capillaries, coated with 2 mg/mL Polybrene; with 100 mM formic acid as BGE; 10 mM formic acid, 20% methanol as SL; 19 kV separation - Native CZE-ESI separations for intact proteins (Figure 4-D): 35-cm long 20 μm i.d., capillary with 100 mM ammonium acetate at pH 7.1 as BGE; 10 mM ammonium acetate at pH 7.1 as SL; 15 kV separation Figure 1: Schematics of the CZE-nanoESI interface and IMS-MS platforms: A) CZE-nanoESI sheath flow interface [1]; B) Multi-pass SLIM-TW-IMS-TOF-MS [6]; C) DT-IMS-qTOF-MS. Intact protein separations and structural characterization using CZE with DT-IMS-qTOF-MS Enhancing sensitivity of CZE peptide separations with a high charge capacity SLIM-TW-IMS-TOF-MS 1 2 3 4 5 6 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 2200000 run1 run2 run3 counts minutes 6 x10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 +ESI EIC(1560.4734-1606.5202) Scan Frag=175.0V 20160501_10uMproteinmixPB0005.d 1 Counts vs. Acquisition Time (min) 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 0 10 0 100000 200000 300000 400000 500000 counts drift time (milliseconds) pSSS0 SpSS0 SSpS0 100 150 200 250 0 50000 100000 150000 200000 250000 counts drift time (milliseconds) pSSS2 SpSS2 SSpS2 0 10 20 0 2000 4000 6000 8000 10000 12000 counts drift time (milliseconds) Peak1 Peak3 A) B) C) D) Figure 2: A) Total ion traces for CZE-MS and CZE-IMS-MS of APLSFRGSLPKSYVK (peak 1) and its singly phosphorylated counterparts (pSSS,sPSS and SSpS) (peaks 2,3). B) Representative IMS spectra of CE peaks 2 and 3. C) IMS spectra of infused phosphopeptide isomers generated with 1.25-m SLIM- IMS MS. D) IMS spectra of infused phosphopeptide isomers separated by the extended serpentine path SLIM-IMS-MS (30.6-m; 2 cycles). • CZE, IMS and MS provide three separation dimensions which can be optimized for specific analytical problems • CZE-IMS-MS enhances the peak capacity of both peptide and intact protein separations, provides a tool for solution versus gas phase conformations studies, and allows investigation into background electrolytes and pH conditions • The electrokinetic sheath flow of the CZE-nanoESI interface supports stable electrospray even without the addition of organic solvent, allowing analysis of native solution proteins and complexes • The increased charge capacity of SLIM devices helps alleviate low injection volume limitations of fast, high electric field CZE separations • High resolution SLIM-IMS separations enhance CZE peak capacity and overcome quantitation limitations due to co-elution of the isomeric species • CZE-nanoESI can be temporarily stopped without incurring sample losses or significant band broadening A) B) C) D-1) D-2) D-3) Figure 3: A) Total ion electropherograms (TIE) of the replicates of fast CZE-IMS-MS of 20 femtomoles of BSA tryptic digest, generated with a high charge capacity SLIM-IMS-MS platform. B) IMS spectrum corresponding to the TIE of BSA digest. C) Individual 0.25 second IMS frames of the CZE peak for the YICDNQDTISSK peptide (M+H) 2+ . C) Figure 4: A) CZE-MS replicates for 10 μM intact proteins (Alpha Lactalbumin, Ubiquitin and Lysozyme) separated in acidic conditions. B) TIE and IMS spectrum for analysis of intact protein standards. C) Extracted CZE-IMS TIE for lysozyme. D) IMS spectra of intact proteins in native conditions: D-1 Ubiquitin, D-2 Carbonic Anhydrase, D-3 Myoglobin. Alpha lactalbumin Ubiquitin Lysozyme Extracted ion trace: A) B) C) 1 • Phosphopeptide isomers partially separate in both CZE and IMS dimensions (Figure 2). • Extended path length (30.6 m) SLIM-IMS helps resolve co-eluting CZE species (Figure 2-D) and can be combined with parked CZE separations to enhance peak capacity A) B) C) • CZE allows protein separation in native states (Figure 4) for correlation with IMS structures 10 20 30 40 0 5000 10000 15000 20000 25000 30000 counts migration time (seconds) run1 run2 run3 CE- nanoESI • A high charge capacity prototype SLIM-TW-IMS-MS is compatible with fast CZE-nanoESI separations. CE peak parking for longer analysis times CE- nanoESI Figure 5: One second CE-MS separation steps (25kV CE voltage, 2kV ESI voltage) followed by 20 second CE voltage parking steps (2kV CE voltage, 2kV ESI voltage). Continuous infusion by CZE-TOF-MS of 5 μM peptide APLSFRGSLPKSYVK. A) Total ion trace B) Extracted ion trace of the (M+H) 3+ peptide ion. • NanoESI sheath flow CE-MS interface enables parking of the CE separation while maintaining a stable electrospray. 0 10 20 30 40 50 60 70 80 90 100 1000000 2000000 3000000 4000000 5000000 6000000 7000000 8000000 counts seconds total ion trace 0 10 20 30 40 50 60 70 80 90 100 0 200000 400000 600000 800000 1000000 counts seconds extracted ion trace B) A)
Transcript of CZE-nanoESI-SLIM-IMS-MS Platforms for Comprehensive ......Figure 2: A) Total ion traces for CZE-MS...
Roza Wojcik, Ian K. Webb, Derek F. Hopkins , Spencer A. Prost, Randolph V. Norheim, Daniel Orton, Sandilya V. B. Garimella, Liulin Deng, Ahmed M. Hamid, Ryan Kelly, Yehia Ibrahim, Erin S. Baker, Richard D. Smith
Introduction
Overview Methods Results
AcknowledgementsPortions of this research were supported by grants from the National Institute of General Medical Sciences (P41 GM103493), NationalInstitute of Environmental Health Sciences of the NIH (R01ES022190) and the Laboratory Directed Research and Development Program atPacific Northwest National Laboratory. This research utilized capabilities developed by the Pan-omics program (funded by the U.S.Department of Energy Office of Biological and Environmental Research Genome Sciences Program) and by the National Institute of Allergyand Infectious Diseases under grant U19AI106772. This work was performed in the W. R. Wiley Environmental Molecular SciencesLaboratory (EMSL), a DOE national scientific user facility at the Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battellefor the DOE under contract DE-AC05-76RL0 1830.
References[1] Wojcik et al, RCMS. 2010; 24: 2554–2560[2] Webb et al, Anal. Chem. 2014, 86, 9169−9176[3] Hamid et al, Anal. Chem. 2015, 87, 11301−11308[4] Garimella et al, Analyst, 2015, 140, 6845–6852[5] TOD 4:10 pm: Smith et al; Ultrahigh IMS resolution using novel traveling wave approaches and long serpentine path length
SLIM modules[6] ThOH 8:50 am Webb, et al; Analysis of Protein Structural Changes with High Resolution Structures for Lossless Ion
Manipulations (SLIM) Ion Mobility-Mass Spectrometry[7] WOD 2:30 pm: Garimella et al; Fundamentals of Ion Dynamics in Structures for Lossless Ion Manipulations (SLIM)
Conclusions
Enhancing peak capacity for peptide separations using extended path length SLIM-TW-IMS-TOF-MS
CONTACT: Roza Wojcik, Ph.D.Biological Sciences DivisionPacific Northwest National LaboratoryE-mail: [email protected]
• Capillary zone electrophoresis (CZE) wascoupled with ion mobility and massspectrometry (IMS-MS) to evaluate advantagesof the three dimensional measurements
• CZE-nanoESI-IMS-MS was used to addressproteomic challenges including:- Ultra-sensitive and efficient separations- Enhancement of peak capacity- Separation & structural characterization of
intact proteins in denatured and nativeconditions
Progress towards comprehensive proteomecharacterization relies on the development ofhigh resolution front end separation techniques,efficient ionization and improved ion transmissionmass spectrometric analysis. In this work wecouple CZE-nanoESI with various IMS platforms,including:• a 1-m drift tube-IMS-MS• a high charge capacity Structures for Lossless
Ion Manipulation (SLIM-IMS) travelling wave(TW) device
• a high resolution SLIM-IMS TW device witha variable path length.
CZE coupled with the nanoESI sheath flowinterface (see Figure 1A) allows for sensitiveseparations of peptides and intact proteins invarious background electrolyte conditions.Coupling CZE-nanoESI with IMS-MS providesopportunities to address the challenges ofproteomic analyses, including enhancement ofpeak capacity and sensitivity, analysis ofisomers, and solution phase/gas phase structuralconformations.
www.omics.pnl.govCareer Opportunities: For openings in the Integrative Omics Group at PNNL please visit http://omics.pnl.gov/careers
CZE-nanoESI-SLIM-IMS-MS Platforms for Comprehensive, Ultrasensitive Proteome Analyses
• CZE separations were performed on an in-house constructed CE system.• The CE-MS interface [1] employed a 15 µm i.d. tapered borosilicate glass
emitter and an electrokinetically pumped sheath liquid with 300 µm betweenthe etched capillary exit and the ESI emitter
• Experimental conditions used were as follows:- CZE-ESI of 1 µM phosphopeptide isomers (Figure 2): 30-cm long, 30
µm i.d., capillary with 200 mM formic acid as background electrolyte(BGE); 10 mM formic acid, 20% methanol as sheath liquid (SL); 19 kVseparation,
- CZE-ESI for 10 µM BSA digest (Figure 3): 28-cm long, 20 µm i.d.capillary with 100 mM acetic acid as BGE and 20 mM acetic acid, 20%methanol as SL; 28 kV separation
- CZE-ESI of intact proteins in acidic condition (Figure 4-A-C): 45 cmlong 30 µm i.d., capillaries, coated with 2 mg/mL Polybrene; with 100 mMformic acid as BGE; 10 mM formic acid, 20% methanol as SL; 19 kVseparation
- Native CZE-ESI separations for intact proteins (Figure 4-D): 35-cmlong 20 µm i.d., capillary with 100 mM ammonium acetate at pH 7.1 asBGE; 10 mM ammonium acetate at pH 7.1 as SL; 15 kV separation
Figure 1: Schematics of the CZE-nanoESI interface and IMS-MS platforms: A) CZE-nanoESI sheath flow interface [1];B) Multi-pass SLIM-TW-IMS-TOF-MS [6]; C) DT-IMS-qTOF-MS.
Intact protein separations and structural characterization using CZE with DT-IMS-qTOF-MS
Enhancing sensitivity of CZE peptide separations with a high charge capacity SLIM-TW-IMS-TOF-MS
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Figure 2: A) Total ion traces for CZE-MS and CZE-IMS-MS of APLSFRGSLPKSYVK (peak 1) and its singly phosphorylated counterparts (pSSS,sPSS andSSpS) (peaks 2,3). B) Representative IMS spectra of CE peaks 2 and 3. C) IMS spectra of infused phosphopeptide isomers generated with 1.25-m SLIM-IMS MS. D) IMS spectra of infused phosphopeptide isomers separated by the extended serpentine path SLIM-IMS-MS (30.6-m; 2 cycles). • CZE, IMS and MS provide three separation dimensions which can be optimized for
specific analytical problems• CZE-IMS-MS enhances the peak capacity of both peptide and intact protein
separations, provides a tool for solution versus gas phase conformations studies,and allows investigation into background electrolytes and pH conditions
• The electrokinetic sheath flow of the CZE-nanoESI interface supports stableelectrospray even without the addition of organic solvent, allowing analysis of nativesolution proteins and complexes
• The increased charge capacity of SLIM devices helps alleviate low injection volumelimitations of fast, high electric field CZE separations
• High resolution SLIM-IMS separations enhance CZE peak capacity and overcomequantitation limitations due to co-elution of the isomeric species
• CZE-nanoESI can be temporarily stopped without incurring sample losses orsignificant band broadening
A) B) C)D-1) D-2) D-3)
Figure 3: A) Total ion electropherograms (TIE) of the replicates of fast CZE-IMS-MS of 20 femtomoles of BSA tryptic digest, generated with a high chargecapacity SLIM-IMS-MS platform. B) IMS spectrum corresponding to the TIE of BSA digest. C) Individual 0.25 second IMS frames of the CZE peak for theYICDNQDTISSK peptide (M+H)2+.
C)
Figure 4: A) CZE-MS replicates for 10 µM intact proteins (Alpha Lactalbumin, Ubiquitin and Lysozyme) separated in acidic conditions. B) TIE and IMS spectrum for analysis of intact protein standards. C) Extracted CZE-IMS TIE for lysozyme. D) IMS spectra of intact proteins in native conditions: D-1 Ubiquitin, D-2 Carbonic Anhydrase, D-3 Myoglobin.
Alpha lactalbuminUbiquitinLysozyme
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• Phosphopeptide isomers partially separate in both CZE and IMS dimensions (Figure 2).• Extended path length (30.6 m) SLIM-IMS helps resolve co-eluting CZE species (Figure 2-D) and can be combined
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• CZE allows protein separation in native states (Figure 4) for correlation with IMS structures
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• A high charge capacity prototype SLIM-TW-IMS-MS is compatible with fast CZE-nanoESI separations.
CE peak parking for longer analysis times
CE-nanoESI
Figure 5: One second CE-MS separation steps (25kV CE voltage, 2kV ESI voltage) followed by 20 second CE voltage parking steps (2kV CE voltage, 2kV ESI voltage). Continuous infusion by CZE-TOF-MS of 5 µM peptide APLSFRGSLPKSYVK. A) Total ion trace B) Extracted ion trace of the (M+H)3+ peptide ion.
• NanoESI sheath flow CE-MS interface enables parking of the CE separation while maintaining a stable electrospray.
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