31 [ Lear n mor e at www.water s.com /bioanalysis ]

Fa S t a n d e F F ec t i v e o P t iM iz at io n o F M rM M e t ho d S F o r l c / M S / M S a na lyS iS o F P e P t i d e S

Joanne Mather, Paul D. Rainville, Erin Chambers, and Robert S. Plumb Waters Corporation, Milford, MA, U.S.

inT roduCT ionPeptide-based biotherapeutic compounds are used in a wide

range of fields including endocrinology, hematology, and

neurology to treat a variety of conditions from acromegaly to

Parkinson’s disease.

Biotherapeutic drugs are often synthetic or modified versions of

an endogenous peptide, designed to mimic or enhance activity of

these naturally-occurring compounds, and they are therefore well-

tolerated by the body. They exhibit high specificity, efficacy, and

low toxicity profiles.1 Bioanalysis must be performed to measure

pharmacokinetics/pharmacodynamics (PK/PD), metabolic fate, and

bioequivalence for regulatory submissions. There are currently

more than 40 peptide drugs on the market as well as more than

400 in late stage clinical trials.2

Peptides have traditionally been analyzed by ligand-binding assays

such as ELISA or RIA, which can be time-consuming and expensive

to develop the antibodies and assays.

LC/MS/MS is a well-understood technique for the analysis of small

molecules; use of the same platform for bioanalysis of peptides

offers:

n Broad dynamic range – 3 to 4 orders of magnitude

n Accuracy

n Universal Method – One analytical technique for many

diverse peptides

n Fast method development

Analytical challenges Determining optimum LC/MS/MS operating conditions for peptide

bioanalysis generally requires some trial and error, which for non-

experts can be time-consuming and daunting. In particular, choosing

the optimum ionization mode has traditionally involved physically

changing the MS source. To further complicate matters, it is common

to see 2+, 3+, 4+, and 5+ charge states, depending on the size of the

peptide. This can make decisions in multiple reaction mode (MRM)

MS more challenging as product ions may appear at higher m/z val-

ues than precursors. Thus a wider m/z range needs to be scanned for

fragment identification. A mass range of up to 2000 amu is required

to have the ability to select higher m/z precursors for fragmentation,

for example, 3+ precursor ions of larger peptides.

In this application note we describe the use of Waters’ innovative

IntelliStart™ Software and its use with the ACQUITY UPLC® and

tandem quadrupole Xevo™ TQ MS systems to rapidly develop an

MRM method for peptides/small proteins, with subsequent incor-

poration into an LC/MS/MS method for separation, detection, and

quantitation.

Figure 1. ACQUITY UPLC and Xevo TQ MS.

eX P eriMenTAl A generalized workflow for the development of a peptide MRM

method in bioanalysis is shown in Figure 2. IntelliStart incorporates

software and hardware that greatly simplify the task of selecting the

best parameters for monitoring peptides.

IntelliStart will automatically:

n Monitor system parameters

n Perform mass calibration and set resolution

n Tune source conditions to each compound

n Generate an MS Method for SIR or MRM

n Check LC/MS system performance

n Enable automated generation of optimized SIR and/or MRM

methods for new compounds

IntelliStart has the ability to select product ions at higher m/z valuesthan precursor ions. It will then optimize and develop the followingparameters3: • Ionization mode: ESI or APCi, positive or negative• MRM transition ions (precursor and product) and/or SIR• Capillary and cone voltage• Desolvation gas flow• Collision energy

IntelliStart allows multiple m/z values to be entered for a compound, e.g., the m/z values for the 3+, 4+, and 5+ charged states (Figure 4).

RUN INTELLISTART

ENTER M/Z VALUES INTO INTELLISTART

CALCULATE M/Z VALUES

It is common to see 2+, 3+, 4+, and 5+ charge states. Depending on the size of the peptide, the possible m/z values for the precursor need to be calculated. The Molecular Mass Calculator in MassLynx Software can be used to automatically calculate the m/z values (Figure 3).

INTELLISTART REPORTAUTOMATICALLY GENERATED

Reports are automatically generated specifying the optimized settingsfor the MRM method. The report also details the product ion spectraalong with cone voltage and collision energy values (Figure 5). These IntelliStart-generated settings are automatically used to create an MS method. The report is saved into a user-specified directory forfuture reference.

CHECK LC/MS

The MS method developed by IntelliStart can be used to evaluate thegenerated MRMs against criteria such as intensity, signal/noise, andspecificity. This can be performed automatically using IntelliStart’sLC/MS System Check.4 BioLynx can also be utilized to confirm theidentity of the product ions generated by IntelliStart.

Figure 2. Workflow for the development of a peptide MRM method.

Figure 3. Molecular Mass Calculator in MassLynx Software.

Setting up IntelliStart to select the optimum conditions for MRM

monitoring of a peptide is easily carried out. Since there can be

many charged states, the first step is to use the Molecular Mass

Calculator embedded within MassLynx™ Software (Figure 3) to

calculate the possible charge state m/z values for a compound.

After entering a compound’s formula, the number of required

charge states is generated, dependent on the peptide’s size. The

calculator will then display the possible m/z values.

The next step is simply to input the name of the compound, the

m/z values, and check the Multiply Charged Parents box in the

IntelliStart setup page (Figure 4).

IntelliStart will now optimize conditions as detailed in Figure 2 and

generate a report (Figure 5) detailing the conditions for the best

MRM for the peptide. The report is automatically saved for future

reference and incorporation into routine analysis.

In this example, compounds are sampled from an on-board vial via

fluidics, so no external syringe driver is required.

Tuning can be carried out in “combined infusion” mode to optimize

the method at an appropriate LC flow rate and solvent composition.

As peptides form different charge states depending on pH, a

thorough investigation of mobile phase pH will allow the optimum

conditions to be generated.

Figure 4. IntelliStart’s Sample Tune browser.

waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

PUBS:

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Job # & CC: WATR9752_B

Job Name: WATR9752_B_Xevo_09_DBmec.indd Date: 12-18-08Live: 7”x10” Smallest Trim: 8.25”x10.875” Largest Trim: 8.5”x11.75” Bleed: 8.875”x12 Page: 1 Rev: 1Stage: mech Release: 12-18-08 DB AD: MM CW: GK PM: DG Scale: 100%Color: 4c Gutter: — Other: COPYRIGHT UPDATED TO 2009 ST: DB

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©2009 Waters Corporation. Waters, The Science of What’s Possible, XEVO, IntelliStart, and ACQUITY UPLC are trademarks of Waters Corporation.

[ REACTION ]

Introducing XEVOTM TQ MS, the new tandem quadrupole mass spectrometer from Waters, featuring IntelliStartTM technology for simplified method development. This automated system solution delivers powerful quantitative capability with very low limits of detection —to the widest range of scientists. All this, along with ACQUITY UPLC® compatibility by design, means you get enhanced performance with dramatically improved speed, productivity, and efficiency. Run more samples per day and more compounds in a single analysis. And make critical, time-sensitive decisions with confidence. Find your answer at www.waters.com/xevo

WHEN ANALYZING COMPLEXSAMPLES WITH XEVO TQ MS,WILL YOU BE MORE IMPRESSEDWITH HOW POWERFUL IT IS?

OR HOW ACCESSIBLE IT IS?

The IntelliStart report details parameters determined by the system.

This report is automatically saved for future reference. IntelliStart

also automatically generates an MS method that can then be

used in routine analysis.tion of mobile phase pH will allow for the

optimum conditions to be generated.

ConClusionsn IntelliStart Software provides an efficient, rapid, reproducible,

and effective approach to developing MRM methods for peptide

assays.

n The software intelligently scans for multiple charge states of

the parent peptide and scans for product ions at higher m/z

values than the parents.

n The Xevo TQ MS allows a wide mass range, up to 2000 Da,

to be scanned for fragment identification of larger peptides.

references

1. Bladon C. Pharmaceutical Chemistry: Therapeutic Aspects of Biomolecules. Wiley and Sons. West Sussex, England, 2002.

2. Bioscience Technology. January, 2002.

3. Rainville PD, Mather J, Plumb RS. Automated Development of MRM Methods for Bioanalytical Assays. Waters Application Note. 2008; 720002569en.

4. Mather J, Rainville PD, Plumb RS, Baynham G. Automated System Check for Bioanalytical Assays: IntelliStart. Waters Application Note. 2008; 720002620en.

Waters and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2009 Waters Corporation. Printed in the U.S.A.October 2009 720003254EN AG-PDF

Figure 5. Section of an IntelliStart report.