Online af˜nity and digestion: A ˚exible and robust tool ... · SumTIC. 4 Online af˜nity and digestion: A ˚exible and robust tool for the characterization and quanti˜cation of

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Online af�nity and digestion:A �exible and robust tool for the characterization and quanti�cation of proteins

ASMS 2014 WP161

David Colquhoun1; Mohamed Nazim Boutaghou1;

Rachel Lieberman1; Brian Feild1; Kevin W. Meyer2;

Scott Kuzdzal1 1Shimadzu Scienti�c Instruments, Columbia, MD; 2Per�nity Biosciences, West Lafayette, IN

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Online af�nity and digestion: A �exible and robust tool for the characterization and quanti�cation of proteins

IntroductionTraditional, manual proteolytic digest and immuno-af�nity sample preparation techniques have been plagued by their lack of qualitative and quantitative reproducibility. This has contributed in part to the lack of implementation of protein biomarkers in quantitative MS-based assays. Online automation of digestion can reduce variability, user error, and improve quantitative results. If coupled with online af�nity capture using immobilized antibodies or af�nity

columns, a unique front-to-end solution can be utilized that allows reproducible sample analysis without time-consuming manual protocols. This work demonstrates the �exibility and utility of a completely online, automated solution for LC-MS/MS sample preparation and peptide analysis using both data dependent and targeted work�ows.

Methods• Online affinity capture was performed using an

immobilized anti-Hb column and eluted directly onto an immobilized enzyme reactor (IMER) and subsequently to reversed phase separation.

• Using variables including time, washing stringency and temperature, we optimized each component of the affinity and online digestion process.

• Protein sequence coverage was analyzed using data dependent acquisition on a Shimadzu LCMS-IT-TOF.

• Peptides were selected and optimized using Skyline. Peptide MRM transitions were monitored using the same Perfinity Workstation coupled to an ultra-fast LCMS-8050.

Work�ow• Import Skyline predicted transitions directly into LabSolutions• Identify the most favorable transitions and optimize source and CID conditions• Output data files directly into Skyline and LabSolutions

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Rapid Digestion: Hemoglobin

Figure 1. Data dependent discovery (DDA) of protein isoforms. Digestion using the Per�nity Workstation (PWS) was optimized for hemoglobin variants (4 minute digestion at 40˚C) and mixtures were analyzed using the LCMS IT-TOF with a top 3 approach on a 15-minute gradient at 0.5 mL/min.Coverage at 5 % protein FDR was approaching 100 %, and isoform peptides were identi�ed.

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Figure 2. Reproducible, accurate quantitative analysis of protein isoforms. Protein isoforms identi�ed via DDA were analyzed using the PWS coupled to an LCMS-8050 using the digestion and af�nity parameters optimized in previous experiments. The sickle and wild type peptides were resolved and quanti�ed using a 5 minute gradient at 0.5 mL/min. CVs for the acquisition were below 8 % for all experiments.

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Figure 3. Rapid immuno-MRM of human IgG. Samples were af�nity captured using a Protein G column, digested and analyzed using a Per�nity Workstation coupled to an LCMS-8050 triple quadrupole. Target peptides and transitions were selected using Skyline; source and CID conditions were optimized manually. A standard curve was generated and 12 peptides from the constant and variable regions of IgG were monitored.

Digestion and Immuno-MRM: IgG• Immuno-MRM incorporates online affinity capture, digestion and LC-MS/MS• Automated using Perfinity Workstation with full method completed in under 1 hour• Identified constant and variable region peptides and optimized using Skyline

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Summary• The Per�nity Workstation facilitated rapid method optimization for af�nity enrichment and digestion• Data-dependent analysis resulted in high (>90 %) sequence coverage for target proteins• The same Workstation was coupled to an LCMS-IT-TOF and then an LCMS-8050, allowing seamless transition from

discovery to targeted analysis• The Per�nity Workstation automated af�nity capture, digestion and separation resulting in rapid analysis times (20 - 60

min) , low CVs (<8%) and sensitive detection.


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For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.

© Shimadzu Corporation, 2014

First Edition: June, 2014

www.shimadzu.com/an/

AcknowledgementsThe authors would like to acknowledge the assistance of the Applications Laboratory team at Shimadzu Scienti�c Instruments with experimental design and execution. We would also like to thank John Schiel (NIST) for the gift of Candidate NIST RM 8670 monoclonal antibody lot #3F1b.


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Online af˜nity and digestion: A ˚exible and robust tool ... · SumTIC. 4 Online af˜nity and digestion: A ˚exible and robust tool for the characterization and quanti˜cation of