Solutions for the Protein LC/MS Laboratory: MassHunter Walkup › cs › library › slide... ·...

Solutions for the Protein LC/MS Laboratory: MassHunter Walkup

Thomas M Trainor, PhD

Account Manager, Life Sciences Group

Agilent Technologies


Common Challenges in Characterizing Biomolecules

Molecular size (insulin is 5 kDa, mAbs are > 100 kDa) Non-enzymatic Post-Translational Modifications can occur during storage or degradation Optical (Circular Dichroism, fluorescence) and Separation (SEC, Analytical Ultracentrifugation) techniques do not provide detailed information on higher-level structure Gels and Western blots may not resolve differences well


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Benefits of LC/MS for Protein Characterization:

MS provides molecular weight confirmation with a high degree of accuracy

MS can validate protein sequences MS identifies post-translational modification (PTM) and its

location via MS/MS MS can be automated for higher productivity and

accessibility

LC/MS offers selectivity and specificity!


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Intact Protein Analysis – the Manual Process


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Data Acquisition

MS Spectrum Deconvolution Zero-charge Mass Spectrum

2*G0F (NGA2F) (+1445.3580)

2*G1F (+1607.5013)

1*G1F/G2F (+3377.1458)

Determine Post Translational Modifications

Compare Observed Mass with Expected

Reporting

Sample Preparation and Submission

Intact Protein Analysis – the Automated Process


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Data Acquisition

MS Spectrum Deconvolution Zero-charge Mass Determination

2*G0F (NGA2F) (+1445.3580)

2*G1F (+1607.5013)

1*G1F/G2F (+3377.1458)

Determine Post Translational Modifications

Delivery of Results

Sequence Matching

Reporting

Upload Sequence

Sample Preparation

Automation for Peptide Mapping


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Data Acquisition

Compound Extraction

Delivery of Results

Sequence Matching Sequence Coverage

Enzymatic Digestion

Upload Sequence

Determine Post- Translational Modifications

2*G0F (NGA2F) (+1445.3580)

2*G1F (+1607.5013)

1*G1F/G2F (+3377.1458)

Reporting

Sample Preparation

MassHunter Walkup with BioConfirm:


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“…offered the possibility to accept [protein] sequences by the end user at the time of sample submission, run through the data analysis and produce an annotated report automatically…we produce hundreds of [protein] sequences a year and that really removes a potential point-of-failure and stress point in our analytical workflow…huge time savings…usage has exploded! We [saved] more than 10 FTE weeks a year.”

http://cnpg.comparenetworks.com/163768-Open-Access-Intact-Protein-LC-MS-in-a-Recombinant-Protein-Laboratory/

...changes intact protein analysis from a service to a tool…you become an enabler for their work instead of a gate-keeper, people really appreciate that…it changes the relationship to them routinely.”

“Open Access Intact Protein LC-MS in a Recombinant Protein Laboratory”

http://cnpg.comparenetworks.com/163768-Open-Access-Intact-Protein-LC-MS-in-a-Recombinant-Protein-Laboratory/

LC System: Agilent 1290 Infinity UHPLC with dual temperature zone oven and 6-column selector valve

Agilent 6530 QToF

1 Protein and 1 peptide reverse phase columns at elevated temperature for standard LC/MS and MS-MS

2 different columns at high temperature for antibody applications

2 additional positions available

Common buffer system (formic acid/acetonitrile/water) but potential for second buffer system on the binary pump

| Open Access LC/MS in recombinant protein production | Eric Fang | 26 September 2013 | 8

Open Access, along with appropriate instrumentation, allows flexibility of application


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The Open Access environment easily saves 5-10 analyst minutes per intact mass sample • Sample preparation and submission are off-loaded onto the end user • Peak extraction and zero-charge mass deconvolution are fully automated • If the user provides a sequence at submission, basic mass matching and results

annotation are provided automatically

System introduced February 1, 2012 with almost 2400 samples processed through the end of year, and on track to double usage in 2013

A busy month in the old environment might have been ~80 samples

| Open Access LC/MS in recombinant protein production | Eric Fang | 26 September 2013 |

Protein Open Access is a success!

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Ease of use and accessibility increase usage

Reduces the burden of sample submission

Reduces the turnaround time to usable results

Increases the overall availability of the resource

Skilled analysts become even more of an enabler, and less of a gatekeeper

Skilled analysts are more free to make contributions in other ways • In the research environment, few have dedicated technical roles

Open Access changes MS from a service to a tool

| Open Access LC/MS in recombinant protein production | Eric Fang | 26 September 2013 | 12

Open Access Protein MS changes the relationship between the user and the technique

3 Step Sample Submission


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Workflows eliminate unnecessary fields and mouse clicks


Page 1: Choose Workflow

Page 2: Fields change to reflect workflow selected - Examples: Formula, Protein Sequence, Modifications, Dilution Factor

New Protein Sequence Editor and Import Formats

• A Protein Sequence Editor program that users can freely install on their desktops and laptops

• Import of FASTA and TXT sequence formats

• Included in Sample Submission in Walkup

Agilent Restricted

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BioConfirm Reports from MassHunter Walkup

Agilent Restricted

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Sample Reprocessing

Common problem is incorrect formula or protein sequence entered, user wants to reprocess without rerunning


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1. Select Reprocess 2. Enter search Criteria 3. Select Samples and

modify input

Benefits of MassHunter Walkup for the Sample Submitter • Access to LC/MS capabilities

without needing to be an expert in LC/MS

• Control over sample preparation and treatment and storage

• Many applications possible - Clone selection - Formulation optimization - Covalent inhibitor studies - Protein ID by Peptide LC-MS/MS - Comparing unmodified and modified

samples (without knowing the sequence)

• Consistent data analysis and spectral deconvolution allows for greater confidence during comparative studies

- Example: pseudo 1st order rate constants


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Benefits of MassHunter Walkup for the Administrator

• View Instrument status • Add/remove/modify users and

update methods from any network computer

• Automatic notification of instrument errors via email or text (SMS)

• Remotely take an instrument offline (e.g., for maintenance)

• Existing queue finishes • No new samples can be submitted

• Facilitated by OpenLAB Shared Services


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Popular add-on hardware for Walkup Systems:


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External Tray (G1313-60004) as extension for walk-up environments Option for vial disposal (G1313-27302)

Add column selection valves, including the new G7116B 1290 Infinity II Multicolumn Thermostat

OpenLAB Shared Services Manage MassHunter and OpenLAB-controlled instruments


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Server

Administrator

Where to Find More Information

Walkup Brochure 5991-3836

Walkup Method Development 5991-2868

Walkup Technical Overview 5991-2868

Application Notes • Intact Protein Analysis Using an Agilent 6560 Q-TOF Mass Spectrometer 5991-2116EN • Analysis of Polyethylene Glycol (PEG) and a Mono and Di-PEGylated Therapeutic Protein Using HPLC and Q-TOF Mass Spectrometry • Reverse Phase Separation of Intact Monoclonal Antibodies Using Agilent ZORBAX Rapid Resolution High Definition 300SB-C3 Column 5900-9667EN • Reverse-Phase Optimization for Ultra-Fast Profiling of Intact and Reduced Monoclonal Antibodies Using ZORBAX Rapid Resolution High Definition

300SB-C3 Column 5990-9667EN • Fast Separation of Recombinant Human Erythropoietin Using Reversed Phased Agilent ZORBAX RRHD 300SB-C18, 1.8 µm 5990-9248EN • Analysis of Oxidized Insulin Chains using Reversed Phase Agilent ZORBAX RRHD 300SB-C18 5990-7988EN • Peptide Mapping of Glycoprotein Erythopoietin by HILIC/MS and RP-LC/MS 5991-2085EN • Rapid UHPLC Analysis of Reduced Monoclonal Antibodies using an Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300SB-C8 Column

5990-9631EN • High Resolution Glycopeptide Mapping of EPO Using an Agilent AvanceBio Peptide Mapping Column 5991-1813EN • Reversed-Phase Separation of Intact Monoclonal Antibodies Using Agilent ZORBAX Rapid Resolution High Definition 300SB-C8 1.8 μm Column

5990-9016EN • Reversed-Phase Optimization for Ultra-Fast Profiling of Intact and Reduced Monoclonal Antibodies Using ZORBAX Rapid Resolution High Definition

300SB-C3 Column. • Ultra High Speed and High Resolution Separations of Reduced and Intact Monoclonal Antibodies with Agilent ZORBAX RRHD Sub-2 μm 300 Diphenyl

UHPLC Column 5990-9668EN • Fast Separation of Recombinant Human Erythropoietin Using Reversed Phased Agilent ZORBAX RRHD 300SB-C18, 1.8 μm 5990-9248EN • Analysis of Oxidized Insulin Chains using Reversed Phase Agilent ZORBAX RRHD300SB-C18 5990-7988EN


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Check out the video:


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APPENDIX


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Where to Find More Information

Walkup Brochure 5991-3836

Walkup Method Development 5991-2868

Walkup Technical Overview 5991-2868

Application Notes • Intact Protein Analysis Using an Agilent 6560 Q-TOF Mass Spectrometer 5991-2116EN • Analysis of Polyethylene Glycol (PEG) and a Mono and Di-PEGylated Therapeutic Protein Using HPLC and Q-TOF Mass Spectrometry • Reverse Phase Separation of Intact Monoclonal Antibodies Using Agilent ZORBAX Rapid Resolution High Definition 300SB-C3 Column 5900-9667EN • Reverse-Phase Optimization for Ultra-Fast Profiling of Intact and Reduced Monoclonal Antibodies Using ZORBAX Rapid Resolution High Definition

300SB-C3 Column 5990-9667EN • Fast Separation of Recombinant Human Erythropoietin Using Reversed Phased Agilent ZORBAX RRHD 300SB-C18, 1.8 µm 5990-9248EN • Analysis of Oxidized Insulin Chains using Reversed Phase Agilent ZORBAX RRHD 300SB-C18 5990-7988EN • Peptide Mapping of Glycoprotein Erythopoietin by HILIC/MS and RP-LC/MS 5991-2085EN • Rapid UHPLC Analysis of Reduced Monoclonal Antibodies using an Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300SB-C8 Column

5990-9631EN • High Resolution Glycopeptide Mapping of EPO Using an Agilent AvanceBio Peptide Mapping Column 5991-1813EN • Reversed-Phase Separation of Intact Monoclonal Antibodies Using Agilent ZORBAX Rapid Resolution High Definition 300SB-C8 1.8 μm Column

5990-9016EN • Reversed-Phase Optimization for Ultra-Fast Profiling of Intact and Reduced Monoclonal Antibodies Using ZORBAX Rapid Resolution High Definition

300SB-C3 Column. • Ultra High Speed and High Resolution Separations of Reduced and Intact Monoclonal Antibodies with Agilent ZORBAX RRHD Sub-2 μm 300 Diphenyl

UHPLC Column 5990-9668EN • Fast Separation of Recombinant Human Erythropoietin Using Reversed Phased Agilent ZORBAX RRHD 300SB-C18, 1.8 μm 5990-9248EN • Analysis of Oxidized Insulin Chains using Reversed Phase Agilent ZORBAX RRHD300SB-C18 5990-7988EN


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More on Information pMod Deconvolution

• Reduction of artifacts

• Spectral data that does not fit the model is rejected as noise

• Improved mass resolution in mixtures

• Uncertainty is represented as peak width


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Classical Maximum Entropy Deconvolution

pMod Deconvolution

New Walkup Administration

May 12, 2015 Walkup Software

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Logical navigation for system setup

Common Tasks on Toolbar

Import/Export Utilities

Administration – One location for Method Setup


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Brings up ChemStation (and ASR) method for reviewing/editing – no need to exit and go to ChemStation

Administration- Validation of all Changes


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Double clicking error takes you to area needing editing

Solutions for the Protein LC/MS Laboratory: MassHunter Walkup › cs › library › slide... ·...

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