MS and MS/MS - swisslabs.eu · Agilent 6520 Accurate-Mass Q-TOF LC/MS The power of MS and MS/MS....

Accurate Mass

Our measure is your success.

Agilent 6520 Accurate-Mass Q-TOF LC/MS

The power of

MS and MS/MS

Redefining Q-TOF performance

The Agilent Technologies 6520 Accurate-Mass Q-TOF LC/MS redefines Q-TOF performance. With innovationslike 32 Gbit/sec data sampling, high-efficiency ion optics, and temperature-stabilized ADC time-of-flightelectronics, the 6520 achieves a combination of mass accuracy and resolution, sensitivity, and in-spectrumdynamic range that far exceeds that of other Q-TOF systems and orbital trapping instruments. The accurate-mass Q-TOF provides the capabilities you need to profile, identify, characterize, and even quantitate samplesas diverse as drug impurities, endogenous metabolites, and protein biomarkers.

Uncompromising performance helpsyou tackle every challenge

The Agilent 6520 Accurate-Mass Q-TOFLC/MS redefines Q-TOF performance with:

• 1–2 ppm MS and 2–4 ppm MS/MS typical mass accuracy

• Enhanced mass resolution—especially at lower masses—with no tradeoff inspectral acquisition rate

• On-column sensitivity in the high-femtogram-to-low-picogram (attomole-to-low-femtomole) range

• Up to 5 orders of in-spectrum dynamic range

• Spectral acquisition rate of up to 10 MS/MS spectra per second

• Broad m/z 25–20,000 TOF mass range

Unlike many competing Q-TOF and orbitaltrapping systems, the 6520 achieves all ofthis without having to significantly compro-mise one aspect of performance to improveanother.

Software turns your data into answers

The accurate-mass Q-TOF includes powerfulMassHunter Workstation software withadvanced data acquisition and data analysiscapabilities, including:

• State-of-the-art feature extraction anddata mining

• Molecular formula confirmation and molecular formula generation

• Deconvolution and accurate intact-proteinmass determination

• Sample-centric quantitation with helpful features like batch-at-a-glancedata review

When necessary, MassHunter software canbe augmented by advanced, application-specific software packages for:

• Differential mass profiling to find potential biomarkers

• Metabolite identification and metabolitedatabase searching

• Protein identification through databasesearching and de novo sequencing

Complete solutions to ensure yourLC/MS success

Agilent can supply you with complete solutions—from sample preparation todata analysis—to turn your samples intovaluable answers.

• Innovative sample preparation technologiessuch as OFFGEL electrophoresis andimmunoaffinity removal of high-abundance plasma proteins

• Industry-leading LC systems for everyflow rate and application, including revolutionary HPLC-Chip/MS technology

• Unparalleled ionization choices including:ESI, APCI, multimode, APPI, and MALDI

• Service and support to make sure your accurate-mass Q-TOF system isalways productive

• Knowledgeable application consulting

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Accurate-massmeasurements forbetter identificationand greater confidence

The 6520 delivers unsurpassed Q-TOFmass accuracy—typicallyas good as 1–2 ppm MSand 2–4 ppm MS/MS—for positive identification.Even under the mostchallenging real-worldconditions, it deliversthe mass accuracy you need for absoluteconfidence in your analytical results.

In contrast to instru-ments that rely on orbitaltrapping, the accurate-mass Q-TOF canacquire many high-quality MS and MS/MSspectra from even narrow 2-second peakswithout compromising its superlative massaccuracy. This fast spectral acquisition minimizes or eliminates undersampling ofcomplex mixtures.

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At the same time, the 6520's fast dataacquisition system provides an in-spectrumdynamic range of up to five orders of magnitude. This facilitates the detectionand identification of low-level compoundsin the presence of much more abundantcompounds. It does so without the sensi-tivity losses caused by beam attenuation in TOF-based instruments with TDC detectors.The dynamic range exceeds that of otherQ-TOFs and that of orbital trapping instru-ments, which are constrained by ion trapcharge capacity limitations.

Digestion of an E. coli sample produced thousands of peptide fragments with a vast range of compositions and concentrations.Using data-dependent acquisition under these extremely challenging conditions, the 6520 accurate-mass Q-TOF produced outstanding mass accuracy for thousands of true positive peptide matches.

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In-Spectrum Dynamic Range

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Maximum theoretical forthis experiment

Erythromycin, 500 fg/µL[M+H]+ = m/z 734.46852Mass error = +0.6 ppmAbundance = ~250 counts

Niacinamide, 10 ng/µL[M+H]+ = m/z 123.05529Mass error = +0.8 ppmAbundance = ~9 x 106 counts

~3 x 107 / 250 = 1.2 x 105

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Mass-to-charge ratio (m/z)734 735 736 737 738 739 740

(m/z)7 740

Analysis of a mixture containing compounds at radically different amounts demonstrates better than four orders of magnitudedynamic range available in the 6520.

Outstanding sensitivity and a wide dynamic range find even trace components

Both sensitivity and dynamic range arecritical for applications such as the identification of low-level impurities or low-abundance proteins. The accurate-mass Q-TOF delivers real-world, on-column sensitivity in the high-femtogram-to-low-picogram (attomole-to-low-femtomole) range.

Unlike instruments that rely on orbital trapping, the 6520 achieves its outstandingmass resolution without sacrificing dataacquisition rates. It can acquire as many as 20 MS spectra per second or 10 MS/MSspectra per second or, more commonly, acombination of many MS and MS/MSspectra each second. Faster spectral acqui-sition makes the 6520 compatible withhigh-throughput applications that demandrapid-resolution chromatography.

Wide mass range for analysis of complex proteomic samples

The 6520 has a broad m/z 25–20,000 TOFmass range that allows it to comfortablydetect both low-molecular-weight com-pounds or fragments, and intact proteins.Orbital trapping instruments have limitationsat both ends of the mass range. They canmiss fragment data critical to de novosequencing and PTM analysis. They can alsohave difficulty with large, intact proteins suchas antibodies.

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Power to resolve very similar masseswithout sacrificing spectral acquisition rates

The 6520 accurate-mass Q-TOF features a32 Gbit/sec data acquisition system thatsignificantly increases mass resolution—especially at lower masses. The outstand-ing resolving power of the 6520 minimizesthe chance that a mass peak of interestwill be hidden by an interfering ion fromthe sample or background. The 6520 letsyou distinguish low-molecular-weight compounds with identical nominal masses.

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Mass-to-charge ratio (m/z)194 194.2 194.4 194.6 194.8 195 195.2 195.4 195.6 195.8 196 196.2

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195 195.1 195.2Mass-to-charge ratio (m/z)

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Methyl 5-acetylsalicylate[M+H]+ = m/z 195.06519Mass error = +0.8 ppm

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Butyl paraben[M+H]+ = m/z 195.10157Mass error = -0.9 ppm

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~14,000 resolution FWHM

6520 analysis easily resolves two compounds with nominal masses of 195 u whose exact masses differ by only 0.03638 u.

Software that helps you more easilyfind answers

The 6520 accurate-mass Q-TOF uses theAgilent MassHunter Workstation software,which provides intuitive yet powerfulinstrument control, data acquisition, andqualitative and quantitative data analysis.MassHunter software includes unique datamining functionality, including powerfulmolecular feature extraction and molecularformula generation algorithms, that enhancesidentification of individual compounds incomplex mixtures.

For analysis of proteins, the MassHuntersoftware includes separate deconvolutionalgorithms individually optimized for peptidesand proteins.

MassHunter software can be enhancedwith optional, application-specific software packages:

• MassHunter Profiling software – quicklyand easily identifies statistically significantvariations between two samples or sample groups

• MassHunter Metabolite ID software –automatically correlates MS/MS spectrafrom precursor drugs and metabolites,generating scores and color-coded results

• METLIN Personal metabolite databasesoftware – supports metabolomicresearch with a searchable database ofover 15,000 endogenous metabolites

• Spectrum Mill proteomics software –quickly and accurately identifies proteinsthrough database searching or de novosequencing, and includes tools for PTM analysis

• GeneSpring MS software – offers a fullrange of powerful tools for statistical profiling and related data mining of verylarge sample sets

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The molecular feature extraction and molecular formula generation capabilities of the MassHunter software simplify the analysis of complex samples. Thisanalysis of a honey sample demonstrates the system's ability to find hundreds of components at all levels, organize the data for easy review, and suggest possible formulas that fit well with the experimental data.

Faster, more confident profiling andidentification of metabolite biomarkers

LC/MS complements GC/MS approachesto achieve comprehensive profiling ofendogenous metabolites. The accurate-massQ-TOF is a highly versatile instrument, perfectfor both profiling metabolites and identifyingany putative biomarkers discovered throughprofiling. For metabolomics researchers, itoffers:

• Stable, reproducible results that minimizethe number of sample replicates required

• Fast spectral acquisition and superiormolecular feature extraction to find morebiomarker candidates

• Superior tools for data comparison andvisualization with optional GeneSpringMS profiling software

• The most complete metabolite databaseavailable in the optional METLINPersonal metabolite database

• Accurate-mass MS and MS/MS data thatincreases the confidence of identifications

Unsurpassed reproducibility minimizes sample requirements

The number of samples that must be analyzed to conclude that a variation is statistically significant depends on bothnatural and analytical variability. Theunsurpassed reproducibility of AgilentLC/MS systems reduces analytical variability,allowing you to minimize the number ofsamples you must analyze duringmetabolite profiling.

Fast spectral acquisition for betterthroughput and data quality

The 6520 can acquire up to 10 MS/MSspectra per second to keep up with high-throughput, rapid-resolution chromatographyand still acquire plenty of high-quality data.

Molecular feature extraction increases the number of metabolites found

Biomarker discovery starts with finding asmany metabolites as possible in your samples. The MassHunter software's uniquemolecular feature extraction algorithmfinds compounds in your chromatograms,even if the compounds do not correspondto obvious chromatographic peaks.

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GeneSpring MS provides superiortools for profiling and visualization

GeneSpring MS software is a powerfulsolution for profiling data from multiplesamples and classes. It includes compre-hensive tools for statistical analysis, data mining, and visualization.

METLIN Personal metabolite databaseaids metabolite identification

After putative metabolite biomarkers havebeen found, they must still be identified.The METLIN Personal metabolite databaseis the most comprehensive metabolitedatabase currently available, with entriesfor over 15,000 endogenous and exogenousmetabolites, as well as di- and tri-peptides.Searching the METLIN database with accurate-mass MS data can greatly reducethe number of possible metabolite identities.This copy of the METLIN database is personal, so you can securely add yourown proprietary compounds for even better searching.

Accurate-mass MS and MS/MSspectra add confidence

Accurate-mass MS precursor data facili-tates more focused database searches,while accurate-mass MS/MS fragmentdata helps you logically confirm or rejectpotential identities provided by a databasesearch. When potential biomarkers are notfound in a database, accurate-mass MSand MS/MS spectra provided by the 6520become even more essential to identifyingyour metabolites.

More confident analysis of lower-molecular-weight compounds

The Agilent 6520 Accurate-Mass Q-TOF LC/MS is an extremely versatile platform for the analysis of lower-molecular-weight compounds. From metabolite profiling to pharmaceutical impurity analysis, fromidentifying drug metabolites to targeted quantitation, the specificity of accurate-mass MS and MS/MSspectra provides results in which you can have the utmost confidence.

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Using accurate-mass data to search the METLIN Personal metabolite database can significantly reduce the number ofpossible metabolite identities.

Evaluation of an MS/MS spectrum for correlation with the structure of the proposed biomarker can be the final step in the identificationof a metabolite biomarker.

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Profile, identify, andquantify impurities

When you are lookingfor impurities such asdegradation productsin stability studies, theaccurate-mass Q-TOFis an all-purpose solution. You can profile your samples,locate and identify theimpurities, and evenquantify both nativecompounds and impurities; all with asingle instrument.

• Fast data acquisition iscompatible with fast chromatography,increasing throughput

• Molecular feature extraction finds morecompounds

• MassHunter Profiling software makes iteasier to pinpoint statistically significantvariations

• Accurate-mass MS and MS/MS spectrafor easier, more confident identification

• Easy relative or absolute quantitation

Fast data acquisition increasesthroughput

Fast, reproducible chromatography with theAgilent 1200 Series RRLC and fast dataacquisition by the 6520 reduce sampleanalysis time, allowing you to process a largenumber of samples without sacrificing dataquality. The unique multimode ion sourcecan further boost productivity by acquiringESI and APCI spectra simultaneously.

MFE does a better job of findingdegradation products

Molecular feature extraction (MFE) doesn'tjust find obvious chromatographic peaks. It analyzes covariant ions to identify asmany compounds as possible in your samples—even when they coelute. Thanksto the wide dynamic range and sensitivityof the 6520, you can find low-abundanceimpurities even in the overwhelming presence of native compounds.

Software tools simplify profiling

MassHunter Profiling software gives youthe visualization tools to identify statisticallymeaningful differences between samples.It calculates abundance ratios and clearlyidentifies differentially expressed features.

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Comparison of amoxicillin and an amoxicillin standard using MassHunter Profiling software reveals two impurities (degradants)tentatively identified as amoxicillin penicillinoic acid (2) and diketopiperazine amoxicillin (3).

Analysis of an MS/MS spectrum from impurity 3 (precursor m/z 366.1126) shows product ions that logically correspond to the structure of diketopiperazine amoxicillin.

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Accurate-mass MS and MS/MS datafor positive identification

If you need to identify impurities, Agilent'smolecular formula generation program cantake advantage of the added specificity ofaccurate-mass MS/MS spectra.

Straightforward quantitation withsoftware that makes it simple

The MassHunter software's quantitationpackage makes it very straightforward toobtain relative or absolute quantitativemeasurements of identified impurities. This software offers parameter-less integration, a curve-fit assistant, dynamicallylinked results, and batch-at-a-glanceresults review.



Streamlined metabolite identification

High-throughput identification of exogenousmetabolites requires a fast, reliable LC/MSsystem with software that simplifies yourtask. The 6520 accurate-mass Q-TOF deliversthe MS and MS/MS data quality of aresearch-grade mass spectrometer withthe reliability and ease of use of a singlequadrupole or TOF instrument. And newMassHunter Metabolite ID software vastlysimplifies metabolite identification.

• Superior accurate-mass MS and MS/MSdata results in better, more accurate identification

• A wide in-spectrum dynamic range andunsurpassed Q-TOF sensitivity help findlow-abundance metabolites in the pres-ence of more abundant compounds

• Unique molecular feature extraction algorithms do a better job of findingmetabolites

• Metabolite ID software increases productivity and throughput

Accurate-mass data improves positive identification of metabolites

Accurate-mass data increases your rate ofpositive identification. The 6520 deliversaccurate mass MS and MS/MS data with-out sacrificing data acquisition speed orsensitivity. It is fully compatible with fast,high-throughput chromatography frequentlyused for metabolite identification.

Wide dynamic range and superiorsensitivity uncover low-abundancemetabolites

TOF and Q-TOF instruments with TDCdetector electronics generally have verynarrow in-spectrum dynamic ranges. Thismakes it difficult or impossible for them tofind lower-abundance compounds in thepresence of higher-abundance compounds.The accurate-mass Q-TOF uses ADC detectorelectronics and a 32 Gbit/sec data acqui-sition system that provide up to 5 orders ofmagnitude of in-spectrum dynamic range.

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MassHunter Metabolite ID software facilitates high-throughput metabolite identification and includes advanced features such as theAuto-Shift algorithm, which considers possible mass shifts caused by biotransformations when determining the final score.

Molecular feature extraction findsmore compounds

The wide in-spectrum dynamic range issupplemented by the MassHunter soft-ware's molecular feature extraction (MFE)algorithm. The result is more metabolitesfound.

Metabolite ID software finds metabolites quickly and easily

Completely new metabolite identificationsoftware uses the well-known Auto-Shiftalgorithm from Novatia to improve metabo-lite identification. The algorithm correlatesMS/MS spectra from the parent drug andfrom your samples and generates a correla-tion score. It considers possible massshifts caused by individual and multiplebiotransformations in determining a finalscore. The color-coded results are easy toreview, and you can accept the results orgo back and adjust the filtering criteria.Results can be exported in Excel format.

Targeted quantitation with the specificity of full-spectrum, accurate-mass data

Q-TOFs are most commonly associated withqualitative analyses, but the specificity ofaccurate-mass measurements and MS/MSspectra also benefits quantitative applica-tions. The versatile 6520 accurate-mass Q-TOF can easily quantitate previouslyidentified metabolite biomarkers and manyother compounds. Advantages of quantita-tion with the 6520 include:

• Greater specificity of accurate-mass, full-spectrum MS and MS/MS

• Target lists ensure the 6520 acquires asmuch data from your target compoundsas possible

• Retention time windows improve thequality of spectra acquired

• Powerful yet easy-to-use quantitationsoftware

Specificity of accurate-mass, full-spectrum MS and MS/MS

The ability of the 6520 to acquire full-spectrum, accurate-mass MS and MS/MSdata lends increased confidence to yourtargeted quantitation. Based on accurate-mass spectra, you can be absolutely sureyou are quantitating the correct compounds.

Targeted-mass lists eliminate data dependence

With the 6520's targeted-mass lists, data areacquired regardless of whether the targetions meet arbitrary abundance thresholdsor are among the n-most abundant ions.This ensures that as much data as possibleis acquired from target compounds.

Retention time windows improvespectral quality

The ability to specify individual retention timewindows for each target ion maximizes thequality of MS/MS spectra. By concentratingon one ion, or only a few ions, in a giventime period, the 6520 can acquire and average more MS/MS spectra from thetarget ions for better sensitivity and spectral quality. This is of particular benefitwhen you are trying to quantitate multiplecompounds in complex samples.

Easy-to-use quantitation software

The MassHunter quantitation softwareincludes numerous features that help youachieve fast, accurate quantitation:

• Choice of integrators including the newparameter-less integrator

• Curve-fit assistant

• Dynamically linked results

• Batch-at-a-glance results review withoutlier flagging

The MassHunter software's quantitation package vastly simplifies quantitation and can perform targeted quantitation using either MS orMS/MS data.

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1. Profiling to find possible biomarkers

2. Identification of the putative biomarkers

3. Characterizing the putative biomarkers

4. Confirmation of biomarker significance



Profile, identify, characterize, and quantify biomolecules—with a single instrument

Quadrupole time-of-flight mass spectrometers are closely associated with protein identification. The combination of accurate-mass measurements and MS/MS spectra make identification easier and moreconfident. The Agilent 6520 Accurate-Mass Q-TOF LC/MS is much more than just an identification solution,however. It can take you from profiling and discovery to identification and characterization, and on throughquantitative confirmation of your proteins of interest.

Discover more protein biomarkers

Protein biomarker discovery is a complexprocess with many analytical challenges. The6520 Q-TOF meets these challenges with:

• The sensitivity and in-spectrum dynamicrange to find more biomarkers

• Software that finds more unique com-pounds and provides more confident andeffective profiling

• Accurate-mass MS and MS/MS structuralinformation for more positive identification

• System-wide reproducibility that reduces the number of false positives and minimizes the number of samplereplicates needed

Better sample preparation yields better analytical results

Agilent's multiple affinity removal columnsand mRP-C18 high-recovery protein columnstogether remove interfering high-abundanceproteins and salts, allowing the unsurpassedsensitivity and in-spectrum dynamic range of the 6520 to find more significantproteins—even at ng/mL concentrations.

Molecular feature extraction locatesmore biomarker candidates

The MassHunter software's unique molec-ular feature extraction algorithm helps youfind more potential biomarkers. By lookingfor covariant ions in chromatograms, itfinds compounds that are present even ifthey are not chromatographically resolved.

Software tools that provide better profiling

MassHunter Profiling software andGeneSpring MS software include thesophisticated statistical comparison andvisualization tools you need to profile twosamples—or hundreds.

Outstanding analytical reproducibilityreduces sample requirements

The excellent reproducibility of HPLC-Chipseparations and 6520 analyses minimizesthe number of sample replicates requiredto conclude that differences between samples or classes are statistically significant. By performing both profilingand identification on the same Q-TOFinstrument, you can further reduce analytical variance.

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The 6520 accurate-mass Q-TOF can play a key role at virtually every step in the biomarker discovery workflow.

Faster, more accurate quantitativeanalysis of labeled proteins

Labeling reagents such as iTRAQ, SILAC,and 18O are widely used for the quantitativeanalysis of proteins. A 6520 accurate-massQ-TOF equipped with HPLC-Chip/MS tech-nology is the ideal platform for quantitativeanalysis of proteins. It offers:

• Exemplary stability, consistency, andreproducibility

• Excellent resolution

• Superlative mass accuracy

• Faster analyses

• Superior software for quantification andidentification

• Easier method development

Exemplary stability reduces RSDs andincreases confidence

The value of labeled quantitation is greatlyenhanced if your LC/MS system producesconsistent results from spectrum to spec-trum and sample to sample. MS/MS in the6520 is extremely stable and ion ratios areimpressively consistent. The result is lowerRSDs or fewer samples needed to achievethe confidence you need.

Mass resolution and accuracy to distinguish tags and interferences

The ability to distinguish betweentags/peptides and interfering ions isessential to obtaining accurate abundanceratios. The accurate-mass Q-TOF's 32 Gbit/sec data acquisition system givesyou significantly better mass resolutionthan ion traps, and unlike orbital trappinginstruments doesn't compromise spectralacquisition rates to achieve it.

Superlative MS/MS mass accuracyfor confident identification

The innovative design of the 6520 Q-TOF'scollision cell and ion optics allows thesame reference mass calibration to beused for MS and MS/MS. The resultingMS/MS mass accuracy—frequently asgood as 2–4 ppm—greatly reduces falsepositive rates during protein databasesearching.

Faster separations, faster analyses

Agilent's revolutionary HPLC-Chip/MStechnology provides separations that arehigher resolution, more reproducible, andfaster than separations by conventionalnanocolumns. You get greater throughputwithout sacrificing chromatographic per-formance. And HPLC-Chips consume lesssample, so you have more available foridentification or further analysis.

Spectrum Mill software takes uniqueadvantage of MS/MS mass accuracy

Spectrum Mill software takes uniqueadvantage of the Q-TOF's MS/MS massaccuracy to narrow the list of possibleidentities and eliminate false positives. Itautomatically calculates—using correctionfactors when necessary—iTRAQ ratios.

Easier instrument setup and methoddevelopment

Most of the 6520 operating parametershave fairly broad optima. This makes itfaster and easier to optimize the instrumentand develop methods. It also makes yourmethods more universal.

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iTRAQ tags at m/z 114, 115, 116, and 117and histidine immonium ion at m/z 110 110.07176

117.11488115.10778-4.5 ppm-4.5 ppm

116.11161111.07262

Mass-to-charge ratio (m/z)109 110 111 112 113 114 115 116 117 118

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114.111583.3 ppm3.3 ppm

0.08 ppm0.08 ppm

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6520 Q-TOF is compati-ble with quantitativestrategies such asiTRAQ and provides excellent mass accuracy and mass resolution.



Analytical power to identify evenlow-abundance proteins

The 6520 accurate-mass Q-TOF delivers the performance you need to accuratelyidentify proteins.

• Accurate-mass measurements of MS andMS/MS ions for improved databasesearch scores and fewer false positives

• Superb MS and MS/MS sensitivity toidentify even low-abundance proteins

• Wide in-spectrum dynamic range thatimproves detection of low-abundancecomponents in the presence of higher-abundance components

• Data-dependent acquisition fast enoughto extract critical MS/MS data from chromatographic peaks

• Fast database searches with fewer falsepositives

Accurate-mass measurements without sacrificing sensitivity oracquisition speed

Orbital trapping mass spectrometers andsome Q-TOF instruments have fundamentallimitations. To achieve accurate-massMS/MS measurements, they have to reducedata acquisition speed and/or sacrificesensitivity. They cannot acquire accurate-mass data at speeds compatible with fastchromatography. In contrast, the 6520 canacquire up to 10 accurate-mass MS/MSspectra per second, fast enough to keep upwith your rapid-resolution or nanoflowchromatography.

Studies have shown that using accurate-mass MS and MS/MS data can dramaticallydecrease the number of false positivematches in database searching. Accurate-mass data is also a huge advantage whena protein is not found in a database andyou need to apply de novo sequencing.

Along with superb mass accuracy at real-world acquisition rates, the 6520 delivers unsurpassed sensitivity and the in-spectrum dynamic range necessary toidentify key lower-abundance proteinseven in the presence of common, higher-abundance proteins.

Comprehensive software for protein identification

Agilent Spectrum Mill software takesadvantage of accurate-mass MS andMS/MS data to make more confident protein identifications. It can be used toidentify proteins through database searchingor de novo sequencing. Spectrum Mill software features:

• Intelligent spectral extraction that makesdatabase searches faster and reduces thenumber of false positives

• MS/MS search modes for both unmodifiedand modified peptides

• Relative quantitation of identified proteins

• Supports ICAT, iTRAQ, and stable-isotopelabeling technologies

MASCOT compatibility

If your laboratory has standardized itsprocesses on the MASCOT protein data-base search engine, there is no need tochange. MassHunter software can exportdata in a format compatible with MASCOT.

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Analysis of an E. colisample demonstrateshow use of accurate-mass MS/MS datalike you get with the6520 accurate-massQ-TOF greatly reducesthe rate of false positivematches from proteindatabase searches.

Precursor mass tolerance = 10 ppm

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Easier identification and characterization of post-translational modifications

A significant percentage of proteins in biological systems have post-translationalmodifications (PTMs) such as phosphoryla-tion or glycosylation, adding additionalchallenge to their identification and characterization. The 6520 accurate-massQ-TOF with available Spectrum Mill softwarehas performance and capabilities you need toidentify and characterize PTMs successfully.

• Stable accurate-mass measurementsallow narrower database search windowsfor fewer false positives

• Unrivaled Q-TOF sensitivity finds morelow-abundance proteins

• Fast, data-dependent MS/MS generatesexcellent coverage for more precise PTM location

• Application-specific software simplifiesthe process and generates higher-quality results

Accurate-mass data reduces falsepositives

Accurate-mass data allows you to narrowyour database search window, reducingthe number of false positive protein identi-fications. Spectrum Mill software can takeadvantage of the sub-5-ppm mass accuracyof the 6520's MS/MS data to furtherincrease the accuracy of your searches.

An especially effective approach is to searchfirst for unmodified proteins and save theresults, then re-search this subset for mod-ifications. The result is faster and producesfewer false positives than a single searchof the entire database with modifications.

The sensitivity necessary to find more proteins

Modified proteins are more likely to bepresent at lower abundances. The excellentdynamic range and superior sensitivity ofthe 6520 help you find lower-abundanceproteins in the presence of higher-abun-dance proteins.

Sensitivity can be further enhanced throughthe addition of HPLC-Chip/MS technology,which both increases chromatographicresolution and enhances sensitivity.

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Q-TOF analysis of beta casein from milk clearly reveals phosphate neutral losses indicative of a tetraphosphopeptide, as well as valuable sequence information.

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MS/MS (precursor m/z 1561.6532)of a beta casein tetraphosphopeptideRELEELNVPGEIVEsLsssEESITR 983.5193

(2.6 ppm)

476.2846(4 ppm)

276.1669(1 ppm) 884.4540

(6.4 ppm)605.3256(0.5 ppm)389.2520

(3.3 ppm) 770.4063(1.2 ppm)

1607.8407(7.5 ppm)

1266.6345(0.7 ppm) 1789.9330

(0.7 ppm)

y1

y4

y16-H3PO4

y6

y5

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y16 y18-H3PO4

b5b14

b16-H3PO4

a8

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b6b11

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b3

b2

[M+2H-4H3PO4]2+

[M+2H-2H3PO4]2+

[M+2H-H3PO4]2+

[M+2H-3H3PO4]2+

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Data-dependent MS/MS providespositive identification

Good sequence coverage is essential toidentifying and locating PTMs. The fastdata-dependent MS/MS of the 6520 Q-TOFprovides excellent sequence coverage for awide range of modified and unmodifiedproteins.

Software that reveals PTM locationsand identities

Proper data analysis is essential to PTMlocation and identification. Spectrum Millsoftware can account for common post-translational modifications during databasesearches or de novo sequencing. It alsoallows you to input custom modifications.Spectrum Mill software takes advantage ofthe superb MS/MS mass accuracy of the6520 to increase the accuracy of both data-base searching and de novo sequencing.

MassHunter software can also export datain a format compatible with the MASCOTprotein database search engine.



More accurate QA/QC of recom-binant antibodies and other proteins

Post-translational modifications and chem-ical degradation can significantly affect theproperties and efficacy of antibodies andother recombinant proteins. Effective qualityassurance and quality control are necessaryto assure protein activity and drug safety.You must be able to both verify identity andcharacterize any changes. The 6520 accurate-mass Q-TOF, combined with Agilent's superior sample preparation and separationtechnology, is an ideal platform for proteinQA/QC, providing:

• Accurate molecular weight determinationof the intact protein

• Better LC separations which improvesequence coverage and hence PTM characterization

• Innovative separation alternatives thatcan enhance coverage

More accurate measurement of intact proteins

Accurate molecular weight determinationof intact proteins is an essential step inQA/QC. The high resolution and massaccuracy of the 6520 improve your abilityto accurately determine the molecularweights of proteins, and dramaticallyincrease the confidence of your QA/QCmeasurements. Agilent's MassHunterWorkstation software provides easy andeffective deconvolution using an algorithmspecifically optimized for MS data fromintact proteins.

Better LC separations improve confirmation of modifications

When you discover modifications in intactproteins, you may want to confirm thenature of the modification through digestionand reanalysis. The outstanding chromato-graphic resolution provided by Agilent 1200

LC systems contributes directly to bettersequence coverage needed to fully charac-terize PTMs. The fast MS/MS acquisition ofthe 6520 also improves sequence coverage.

Innovative separation alternatives

Agilent's leadership in separation technologygives you alternatives that can enhanceQA/QC of recombinant proteins. Our uniqueOFFGEL electrophoresis technology sepa-rates glycosylated and phosphorylated proteins based on pI changes. This canimprove subsequent LC separations andhence sequence coverage.

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2647.78339

1756.20662

Mass-to-charge ratio (m/z)

600 1000 1400 1800 2200 2600 3000 3400 3800 4200 4600 5000

Mass spectrum of an intact antibody

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Deconvoluted spectrum

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Deconvoluted mass (u)

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2650.66097

2644.74603

2653.49793

2656.28485

Mass-to-charge-ratio (m/z)2635 2640 2645 2650 2655 2660 2665

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Analysis of an intact antibody demonstrates the 6520's superb mass accuracy and its ability to resolve different glycospecies within the antibody.

Service and support

Agilent Technologies has earned its reputation for one of the finest service andsupport organizations in the life scienceand chemical analysis industries. Oursophisticated communications technologyand infrastructure enable us to operateseamlessly in more than 40 countries, sowe can support you wherever you are. Yet our global perspective does not stop us from being responsive down to the lastlocal detail.

For more information

Learn more:www.agilent.com/chem/qtof

Buy online:www.agilent.com/chem/store

Find an Agilent customer center inyour country:www.agilent.com/chem/contactus

U.S. and [email protected]

[email protected]

[email protected]

Research use only. Information, descriptions and speci-fications are subject to change without notice.

Agilent Technologies shall not be liable for errors con-tained herein or for incidental or consequential dam-ages in connection with the furnishing, performance oruse of this material.

© Agilent Technologies, Inc. 2007Printed in the USA July 31, 20075989-6826EN

MS and MS/MS - swisslabs.eu · Agilent 6520 Accurate-Mass Q-TOF LC/MS The power of MS and MS/MS....

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