A New erA iN ProteiN QuANtificAtioN

[ sYsTEMs BIOLOGY sOLuTIOns ]

MORE PEPTIDES. MORE PROTEINS.

MORE REPRODUCIBILITY.

MORE QUANTIFICATION.

the waters® expressione High Definition Proteomics™ System defines the new

standard for rigorous protein quantification in bottom-up analysis. Designed for

comprehensive quantitative and qualitative protein profiling, this total system

solution combines high bandwidth uPLc/MSe data acquisition and waters strin-

gent protein iD technology – identitye – with multi-peptide, label-free, protein

quantification. Now life scientists can reproducibly report the maximum number

of secure protein identifications together with stringent differential quantification

and absolute protein concentration. the expressione System comprehensively

maps complex biological systems with greater statistical rigor than established

MS-based strategies – enabling a new era in biomedical research.

[ sYsTEMs BIOLOGY sOLuTIOns ]

EXPRESSION

The Waters ExpressionE High Definition Proteomics System integrates Waters nanoACQUITY UPLC® System with a Waters Q-Tof Premier™ or Waters SYNAPT™ High Definition Mass Spectrometry™ System with optimized sample preparation chemistries, statistically rigorous bioinformatics, optimized analytical methods, and comprehensive system training.

[ sYsTEMs BIOLOGY sOLuTIOns ]

t He wAt erS eX P reSSioN e HiGH DefiNit ioN P rot eoMicS SYSt eM

The ExpressionE System has been specifically developed to

address the analytical challenges inherent in contemporary

proteomics including:

n Biomarker discovery

n Molecular diagnostics R&D

n Identifying the component proteins in pull-down experiments

n Protein flux analysis

n Global protein profiling

The Waters ExpressionE High Definition Proteomics System integrates

a Waters nanoACQUITY UPLC System with a Waters Q-Tof Premier

or Waters SYNAPT High Definition Mass Spectrometry (HDMS™)

System with optimized sample preparation chemistries, statistically

rigorous bioinformatics, optimized analytical methods, and compre-

hensive system training. The result is a sophisticated system

solution that facilitates stringent proteomics discovery research

with defined performance.

Meet iNG t He P rot eoMicS cHALLeNGe

Proteomics is increasingly focused on biomarker discovery and

understanding disease mechanisms at the molecular level to

develop molecular diagnostics, targeted therapies, and personalized

medicine. This necessitates identifying the components of complex

mixtures that contain thousands of proteins, and quantitatively

comparing them over a wide dynamic range.

Traditional bottom-up proteomics analysis is frustrated by analytical

incompleteness or under-sampling. This phenomenon is a systemic

weakness of all LC/MS/MS systems operated in the serial, data

directed analysis (DDA) mode. In contrast, the ExpressionE System

employs a unique high bandwidth UPLC/MSE data acquisition strategy

to consistently over sample complex peptide digests to deliver global

ExpressionE datasets containing evidence for all peptides above the

limit of detection.

In differential proteomics (relative quantification), most contempo-

rary approaches necessitate isotopic/isobaric labeling and analysis

of a minority of (affinity selected) peptides associated with each

protein contained within a sample. Conversely, the ExpressionE

System does not require peptide derivatization to quantify

the majority of tryptic peptides within a complex digest.

“i told my students, ‘You don’t know how bad ion

trap (proteomics) data can be – until you have

seen how good expressione data is!’ ”

A leading uS academic and owner of four ion traps

DAtA StriNGeNcY

“in the last ten years, the

field of proteomics has

expanded at a rapid rate.

A range of exiting new tech-

nologies has been devel-

oped and enthusiastically

applied to an enormous

variety of biological ques-

tions. However, the degree

of stringency required in

proteomic data generation

and analysis appears to

have been underestimated.

As a result, there are likely

to be numerous published

findings that are of

questionable quality.”

M.R. Wilkins et al. “Guidelines for the next 10 years of Proteomics.”

Proteomics. 2006 Jan; 6 (1): 4-8.

Go BeYoND DAtA-Direct eD ANALYSiS

As with any analytical technique, the validity of results are constrained by the quality of raw data.

The Waters ExpressionE system combines UltraPerformance LC® with MSE detection and industry-

leading dynamic mass resolution. Waters UPLC/MSE technology consistently over-samples complex

digests for comprehensive peptide detection. This analytically-complete approach delivers protein

identification with unmatched sequence coverage.

Waters’ chromatographic advancements in UPLC, with rugged sub-2 µm Bridged Ethyl Hybrid (BEH)

particle columns, represent the most technologically advanced solution for nanoscale separations

of complex tryptic digests. UPLC enables separations with maximum resolution, speed, and

sensitivity. The peak capacity and robustness of UPLC accentuates the specificity of the ExpressionE

system’s retention time prediction model, allowing the amino acid composition of peptides,

identified from matching MSE spectra, to be corroborated.

Likewise, Waters’ innovations in data-independent MSE have produced a revolutionary data

acquisition technique. Waters’ premier orthogonal time-of-flight (TOF) mass spectrometers provide

the highest dynamic mass resolution for LC/MS applications – with 17,500 FWHM at 10 spectra/

sec – facilitating outstanding peak definition and accurate mass measurement.

UPLC/MSE acquires data with two rapidly alternating MS functions; the first contains exclusively

low-energy mass spectra, the second is composed of mass spectra acquired at elevated collision

energy (designated as MSE). The resulting data set contains a comprehensive time-resolved

record of all detectible precursor and product ions. Precursor and product ions are associated by

both retention time alignment and peak shape, enabling even chimeric peptides and overlapping

ion clusters to be successfully handled. The resulting time-aligned precursor and product ion

mass lists are used for peptide/protein identification with Waters IdentityE bioinformatics. The

intensity of each peptide’s (multiply charged) molecular ions are used for both relative and

absolute quantification.

Data acquisition using UPLC/MSE.

[ E X PRE ssIOn E sYsTEM ]

GLoBAL eX P reSSioN e DAtA SetSGlobal ExpressionE data sets provide a comprehensive record of all peptides detectable by

ESI LC/MS within a sample and can be mined exhaustively for both qualitative and quantitative

information, such as up- and down-regulated proteins, biomarkers, and the abundance of targeted

proteins or peptides. Exact mass retention time (EMRT) signature files can be exported to public

domain data repositories and third-party data analysis packages such as Rosetta Elucidator, Umet-

rics Simca-P or Spotfire, for orthogonal data correlation.

St riNGeNt DiffereNt iAL QuANt if icAt ioNMultiple normalized global ExpressionE data sets are compared and contrasted in relative

protein expression analyses. In all cases a minimum of three technical replicates are analyzed to

facilitate statistically rigorous reporting of quantitative and qualitative results. EMRT signatures

enable all detected peptides to be uniquely identified and enable any two global ExpressionE

data sets to be differentially compared. The normalized intensity of each EMRT signature is

indicative of the abundance of a specific, tryptic peptide. Each protein in a global expression

data set is represented by multiple peptides. The relative abundance of any protein is calculated

by comparing multiply-matched EMRT signatures (n≥3), allowing limits of confidence to

be determined.

Hi3 ABSoLut e QuANt if icAt ioNAbsolute protein quantification conventionally requires co-determination of a unique peptide

for each targeted protein, with its corresponding stable isotope labelled internal standard. Such

isotope dilution methods remain the gold standard for absolute protein quantification and are

essential in clinical diagnostic procedures. However, Waters high-three (Hi3) absolute quantifica-

tion provides a rapid and cost effective intermediate strategy that enables pre-clinical trial

validation of putative biomarkers.

Hi3 methodology references the combined intensity of the

multiply charged molecular ions for the three most abundant

tryptic peptides of a quantitatively added ExpressionE

protein standard with the observed Hi3 response for any

identified protein in a complex digest mixture.

The Hi3 peptide response for any protein is similar;

therefore, the summed intensity of the Hi3 ExpressionE

reference peptides may be used to estimate the molar

amount of any protein present in a complex mixture.

As a result, the Hi3 methodology is applicable to estimate

the absolute concentration of any protein identified within

an ExpressionE data set.

A NeeD for cHANGe

“too many papers that i

receive and/or read in

scientific journals have no

evidence of any repeatability,

much less reproducibility…

thus, i suspect that > 50%

of papers now being published

in the proteomics field have

data that was performed but

once (n = 1), which makes

the entire work unacceptable

for publication.”

W.S. Hancock (Editor-In-Chief). “An Analytical Chemist’s Perspective.” Journal of Proteome Research. 2007 Vol. 6 (5), 1633.

[ sYsTEMs BIOLOGY sOLuTIOns ]

Average Hi3 determined concentration (blue circles) and %RSD (blue whiskers) for 11 human serum proteins from 10 volunteers. Average expected concentrations (red circles) with minimum/maximum values (red whiskers), calculated using both ELISA and enzymatic assay data (Specialty Laboratories). The experimental Hi3 data show a high degree of similarity.

Human Serum Proteins

Hi3 Label-free Absolute Quantification of Human Serum Proteins

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Human Serum Proteins

MrM Met HoDS MADe eASYWaters Global ExpressionE data sets contain a comprehensive

record of masses, charge states, ion intensities, and retention times

of all precursor and fragment ions above the limit of detection.

They contain all the necessary data to construct Multiple Reaction

Monitoring (MRM) methods for high throughput target peptide

analyses on triple quadrupole instruments. Optimum MRM transi-

tions for unique peptides can be rapidly identified and incorporated

into LC/MS/MS methods. From here, they are ported directly to

one of Waters high performance tandem quadrupole MS systems,

which share the same MassLynx™ Software, Z-Spray™ ion source,

and UPLC technologies as the Waters ExpressionE High Definition

Proteomics System.

SYSt eM coMPoNeNtS AND oPt iMiZeD PerforMANc e K itThe Waters ExpressionE System consists of:n Waters nanoACQUITY UPLC Systemn Waters SYNAPT HDMS System n Waters Q-Tof Premier Mass Spectrometern Waters ExpressionE System Informaticsn Waters ExpressionE Applications Kitn Waters ExpressionE System Manual and Documentationn Waters ExpressionE System Training Course

The ExpressionE High Definition Proteomics System

Applications Kit provides all of the materials required

to easily perform the system performance verification

and acceptance test. The kit includes ExpressionE System

Standards, RapiGest™ SF Digest Reagent, nanoACQUITY

UPLC BEH 1.7 µm columns, standard operating procedures,

method templates, and reference data.

[ E X PRE ssIOn E sYsTEM ]

COMPLETENESS

“ Analytical incompleteness refers to a phenomenon where a technique used for the analysis of complex mixtures

of peptides may only yield information for a fraction of relevant peptides in an single analytical run.”

M.R. Wilkins, et al. “Guidelines for the Next 10 Years of Proteomics.” Proteomics. 2006 Jan; 6 (1): 4 - 8.

expressione: Label-free relative Quantification

PROTEINTRUE

FOLD CHANGEEXPRESSIONE FOLD CHANGE

EXPRESSIONE FOLD CHANGE % ERROR

Bovine Serum Albumin 0.00 0.0 0.0 %

Lactoperoxidase 0.00 +0.06 6.0 %

Ribonuclease 0.00 -0.06 6.0 %

Peroxidase C1A 0.00 +0.05 5.0 %

Casein +4.00 +3.89 2.75%

Catalase +5.00 +4.83 3.4 %

Carbonic Anhydrase -3.22 -3.22 0.0 %

Glycogen Phosphorylase -76.9 -66.6 13.4 %

Average = 4.6 %

The fold change differences across two samples containing eight well charac-terized proteins in known amounts was determined using Waters ExpressionE

Label-Free relative quantification protocol with an average error of 4.6%. These data compare very favorably with a recent 20-laboratory study of

identical samples, employing all of the common commercial isotopic/iso-baric labelling techniques, which exhibited an average error of 46%.

tHe uLt iMAt e Prot eoMicS PLAt forM

The ExpressionE High Definition

Proteomics System quantitatively

and qualitatively maps complex

biological digests with greater

statistical rigor than established

MS-based strategies, providing

a secure foundation for biological

discovery and medical research.

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waters corporation 34 Maple StreetMilford, MA 01757 U.S.A.T: 508 478 2000F: 508 872 1990www.waters.com Waters, nanoACQUITY UPLC, UltraPerformance LC, and UPLC are registered trademarks

of Waters Corporation. The Science of What’s Possible, ExpressionE High Definition Proteomics, Q-Tof Premier, High Definition Mass Spectrometry, HDMS, MassLynx, Z-Spray, and RapiGest are trademarks of Waters Corporation. All other trademarks are property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A. January 2008 720002324EN TL-AC

For more information on the Waters IdentityE approach, please refer to the IdentityE High Definition Proteomics System brochure, available on www.waters.com (Literature Code 720002176EN).