Utilizing a Hybrid Mass Spectrometer to Enable Fundamental Protein Characterization:Intact Mass Analysis and Top-DownFragmentation with the LTQ Orbitrap MSTonya Pekar Second, Vlad Zabrouskov, Thermo Fisher Scientific, San Jose, CA, USAAlexander Makarov, Thermo Fisher Scientific, Bremen, Germany

Introduction

A fundamental stage in protein characterization is todetermine and verify the intact state of the macromolecule.This is often accomplished through the use of mass spectrometry (MS) to first detect and measure the molecularmass. Beyond confirmation of intact mass, the next objectiveis the verification of its primary structure, the amino acidsequence of the protein. Traditionally, a map of themacromolecule is reconstructed from matching masses ofpeptide fragments produced through external enzymaticdigestion of the protein to masses calculated from an insilico digest of the target protein sequence. A more directapproach involves top-down MS/MS of the intact proteinmolecular ion.

To accommodate progressive stages of characterization,the choice of instrument for MS-based protocols shouldconsider the diversity of required analyses, including furtherinterrogation of protein sequence modifications, which canbe very labile (glycosylation, phosphorylation). A hybridlinear ion trap-Orbitrap mass spectrometer offers extendedversatility for this application, featuring both high-performancedetection (high resolution, mass accuracy, and dynamicrange) and distinctive flexibility in operation.1-6 It offersseveral choices of fragmentation, to permit sophisticatedexperiments such as post-translational modification (PTM)analysis with preservation of labile modifications (electrontransfer dissociation, ETD),3-35 de novo sequencing (highmass accuracy and combined activation types),36-39 anddisulfide mapping to augment superior, sensitive MS/MSdetection for peptide identification.40-48

This note describes protein characterization using theThermo Scientific LTQ Orbitrap XL or LTQ OrbitrapVelos hybrid mass spectrometer, demonstrating intact proteinand top-down fragmentation analysis (Figure 1).48-58

Experimental

Protein standards, including bovine carbonic anhydrase,yeast enolase, bovine transferrin and human monoclonalIgG, were purchased from Sigma-Aldrich. For direct infusion, proteins in solution were purified by either aThermo Scientific Vivaspin centrifugal spin column or asize-exclusion column (GE Healthcare), employing at leasttwo rounds of buffer exchange into 10 mM ammoniumacetate. Protein solutions were at a concentration of least1 mg/mL prior to clean-up. Samples were diluted into50:50:0.1 acetonitrile:water:formic acid prior to infusioninto the mass spectrometer. Instrument parameters werealtered during infusion of protein solutions to optimize thesignal-to-noise ratio and the accuracy of measurement. ForLC/MS analysis, a polystyrene di-vinyl benzene (PSDVB)protein microtrap (Michrom Bioresources) was utilized todesalt proteins on-line.

Experiments were performed on LTQ Orbitrap XL™

and LTQ Orbitrap Velos™ hybrid mass spectrometers.Top-down experiments were conducted using direct infusionof a 5 pmol/uL solution of enolase. All data were acquiredwith external mass calibration. The Xtract feature withinThermo Scientific Xcalibur software was used to deconvoluteisotopically resolved spectra to produce a monoisotopicmass peak list for high-resolution MS and MS/MS data.Thermo Scientific ProMass Deconvolution software wasused to deconvolute intact protein spectra that containedunresolved isotopic clusters in production of an averagezero charge mass of the intact protein. Thermo ScientificProSightPC 2.0 software was used to map top-down frag-mentation spectral data to associated sequences utilizingthe single-protein mode search. Deconvoluted spectra produced from the Xtract algorithm within Xcalibur soft-ware were imported into ProSightPC software. To accountfor errors in the determination of the monoisotopic mass,a parameter tolerance of 1.01 Da was used to match frag-ments. However, only those fragments which correspond-ed to a mass error of 1.0076 Da ± 5 ppm were consideredcorrect.

Key Words

• LTQ Orbitrap Velos

• LTQ Orbitrap XL

• AppliedFragmentationTechniques

• Electron TransferDissociation ETD

• Top-DownProteomics

ApplicationNote: 498

Figure 1: Instrumental schematic of the LTQ Orbitrap XL ETD massspectrometer, indicating the available fragmentation mechanisms

Results and Discussion

Sample Preparation

Sample preparation prior to electrospray ionization (ESI)is critical for the generation of usable mass spectra, especially for intact protein analysis. A high concentrationof salt or other adducts can suppress ionization and there-fore sensitivity of analysis. It can also complicate spectra,broadening the observed MS peak to reduce sensitivitythrough division of analyte signal among ions of heteroge-neous composition.

Sample purification can be accomplished through bothon-line LC/MS chromatographic separation from salt oroff-line clean-up followed by direct infusion. For proteinmixtures, proteins should be chromatographically resolvedas much as possible to maximize MS detection of individualcompounds. It is optional to use formic acid as a modifierfor reverse-phase (RP) separations to avoid ionization suppression induced by trifluoroacetic acid (TFA), thoughTFA can also improve the concentration of protein elutionduring chromatographic separation which can improvesignal to noise for LC/MS separations.

Mass Spectrometry – Intact Analysis

The mass spectrum of large macromolecules is a compositeof a distribution of naturally-occurring isotopes. As moleculesincrease in mass, the contribution of higher mass isotopes(13C) increases proportionally. For large proteins, the naturaldistribution of these isotopes is nearly Gaussian, with themost abundant isotopes adjacent to the average mass of theprotein. Electrospray ionization (ESI) produces a series ofmultiply-charged ions detectable by the mass spectrometer(Figure 2). Depending on the specification of the massspectrometer, the isotopes of each charge state may or maynot be resolved. The LTQ Orbitrap XL or LTQ OrbitrapVelos hybrid mass spectrometer is capable of resolvingpower in excess of 100,000 (FWHM) at m/z 400. Thisallows isotopic resolution of proteins close to 50,000 Da.

For best analysis of large macromolecules, it wasdetermined that the initial portion of the FT transient produced during image current detection (containing firstbeat) contained the largest signal for transformation oflarge macromolecules with Orbitrap analyzer detection. Thisnecessitates a reduction in the delay between excitation of the ion population (injection of ion cloud into theOrbitrap mass analyzer) and detection of the transient. To accomplish this on LTQ Orbitrap, LTQ Orbitrap XL,and LTQ Orbitrap Discovery mass spectrometers, a toggle

for analysis of high-molecular-weight molecules is accessiblefrom the instrument controlwindow (Tune page) in instru-ment control software version2.5.5 SP1 or later. The toggle islocated under the diagnosticsfile menu under Tools→Toggles→ FT detection delay,and should be set to High forlarge macromolecules. Nochanges are necessary for theLTQ Orbitrap Velos instru-ment.

If isotopic resolution of aprotein is desired and achiev-able, a higher resolution settingshould be used for detection –60,000 or 100,000 resolvingpower. It is recommended thatthe pressure in the Orbitrapanalyzer be less than 2¥10-10

Torr. For larger proteins forwhich isotopic resolution ispossible, it is recommendedthat sufficient spectral averag-ing is conducted to produce thebest isotopic envelope, espe-cially for broadband acquisi-tion.

Figure 2: Mass spectrum of bovine carbonic anhydrase with one acetylation modification (29006.682 Da), depictingisotopic resolution of the z = 34 charge state. Data was acquired at 100,000 resolving power with Orbitrap analyz-er detection. Accurate mass of the most abundant isotope is 1.73 ppm. HCD gas was turned off for acquisition onthe LTQ Orbitrap XL instrument. Source voltage of 10 V was applied.

Alternatively, a subset of charge states can be isolated in the ion trap and subjected to Orbitrapanalyzer detection to increase ion statistics for production of a better quality isotopic envelope,potentially requiring less spectral averaging. Figure 3depicts the mass spectrum of the z = 45 chargestate of yeast enolase with isotopic resolution. Forproteins for which isotopic resolution cannot beachieved (i.e. proteins >50 kDa), it is recommendedto run at the 7500 or 15,000 resolution setting ofthe Orbitrap analyzer due to the interference effectsas described in Makarov et al.53 This results in abetter signal-to-noise ratio and, because of the higherscan rate, allows for more scans averaged across achromatographic peak (Figure 4). The accuracy ofmass measurement for unresolved isotopic clustersis not dependent on the resolution setting.

It was also found that S/N is improved whenthe supply of external gas to the HCD collision cell isreduced. The collision gas can be turned off in thediagnostics file menu within the instrument control

Figure 3: Mass spectrum from LTQ Orbitrap XL mass spectrometeryeast enolase (monoisotopic mass of 46642.214 Da) with broadbandacquisition, depicting isotopic resolution. Inset depicts the z = 45charge state with an accurate mass of 0.12 ppm for the most abundant isotope. Data was acquired at 100,000 resolving powerwith broadband Orbitrap analyzer detection (m/z 900-1200 shown),averaging ~600 scans. HCD gas was turned off for acquisition withthe LTQ Orbitrap XL instrument. A tube lens of 110 V (LTQ OrbitrapXL instrument) or S-lens of 50% (LTQ Orbitrap Velos instrument)was used in combination with 10 V of source voltage.

Figure 4: Mass spectrum of enolase acquired at 7500 resolving power. The spectrum is an average of ~100 scans across a chromatographic peak of 2 µg of enolase injected on a protein microtrap. Mass accuracy of the deconvoluted average mass (46670.6879 experimental) is 2.4 ppm. HCD gas was turned off for acquisition with the LTQ Orbitrap XL instrument. A tube lens of 110 V (LTQ Orbitrap XL instrument) or S-lens of50% (LTQ Orbitrap Velos instrument) was used in combination with 10 V of source voltage.

window (tune software), under Tools→ Toggles→ FTHCD collision gas. The toggle should be set to “off”. Thistoggle is available for the LTQ Orbitrap XL mass spec-trometer. Due to a different layout of gas delivery, theLTQ Orbitrap Velos mass spectrometer offers a lowerOrbitrap analytical pressure than previous systems, andthe HCD gas does not need to be turned off.

As discussed previously, mass spectral protein signal isoften complicated by the association of non-covalentadducts such as salt, acid and solvent adducts. It is bestpractice to achieve efficient desolvation of the proteinupon electrospray ionization in addition to separationfrom salt. The sheath and auxiliary gas in the ESI source inconjunction with increased temperatures (capillary tem-perature, heated electrospray probe) aid in desolvation.These values should be set appropriate to the LC flowrate, and can be tuned manually with infusion of the ana-lyte into the desired LC flow rate. During infusion of pro-tein solutions for optimization of acquisition parameters,it is recommended that the number of microscans beincreased to 5 or more for the full scan. Tuning of thesource parameters should be done using the ion trapdetector.

The application of source voltage (SID) can also beused to aid in desolvation or removal of undesiredadducts. The amount of source voltage to apply is dependenton the size and stability of the protein and also on thestrength of attraction between protein ions and adducts. A setting of 10-20 V is a good starting point, and can be

increased upwards to promote a cleaner signal. For largemacromolecules such as intact antibodies (~150 kDa), asource voltage of >35 V is recommended. It should benoted that the fragility of protein macromolecules andcovalent modifications can vary. Too much energy deposi-tion can also result in loss of fragile modifications or infragmentation of the parent molecule.

Some adjustment of parameters, such as reduction ofsource voltage, temperature, or tube lens/S-lens voltage, maybe required to minimize fragmentation. Alternatively, if aprotein spectrum is pure, a high source voltage can be usedto fragment the intact molecular ion (top-down analysis).This approach has the advantage of subjecting all chargestates to fragmentation, which improves signal for theMS/MS spectrum, as the sum of signal from all contributingcharge states is utilized. This also permits access to alternativefragmentation pathways from different charge states. Asource voltage of up to 100 V can be applied to inducefragmentation of all ions and all charge states.

The voltage of the tube lens in LTQ Orbitrap XL orDiscovery instruments, and the S-lens in LTQ OrbitrapVelos instruments affects ion transmission to the massanalyzer within a desired mass range. For proteins whichionize at higher m/z, a higher lens voltage is required.These values can be adjusted manually. For large macro-molecules such as antibodies, the required voltage can bein excess of 200 V (tube lens) or 80% (S lens). Other ionoptic voltages were found to be much less influential.Deviation from default values influenced signal less than

30% for the proteins analyzed inthese experiments. It is recommendedthat they be left at default or as setduring tuning with a small protein(ex: myoglobin).

For molecules such as intact antibodies analyzed in the high-massrange (up to m/z 4000), an additionalcalibration step is required in order toachieve proper mass accuracy withOrbitrap analyzer detection. Thisadjusts the ion population to thedesired target when ions are ejectedfrom the linear ion trap to the FTMSin high-mass mode, and does so byadjusting the calculated injectiontime. This procedure is also located indiagnostics, under Toggles → Systemevaluation → FT high mass range targetcompensation. Figure 5 depicts themass spectrum for an immunoglobulin(IgG), with associated acquisitionparameters listed. Again, a higher tubelens or S-lens voltage also promotestransmission of higher m/z ions foranalysis in the high-mass range.

Figure 5: Mass spectrum of intact immunoglobulin (IgG, 147,251 Da) detected at 7500 RP by LC/MS.Mass accuracy of the average mass was 6 ppm. A tube lens of 230 V (LTQ Orbitrap XL instrument) or S-lens 90% (LTQ Orbitrap Velos instrument) was used. Source voltage of 40 V was used to removeadducts. HCD gas was turned off for the LTQ Orbitrap XL mass spectrometer.

Mass Spectrometry – Top-Down Analysis

Beyond routine intact mass verification lies the need todetermine or confirm the identity of a protein’s primaryamino acid sequence. As described, the hybrid linear iontrap – Orbitrap system allows multiple activation strategies,which often provide complimentary information. Thehybrid system can also resolve complex fragmentationspectra from high-molecular-weight precursors, whichcontain many fragments of large mass.

Whole protein fragmentation has the advantage of circumventing the limitations of traditional peptide mapping(such as in vitro-induced modifications,

Electron transfer dissociation (ETD) is a kinetic reactionwith the rate dependent on the charge of the precursor –the higher the charge state, the faster the reaction occurs.Shorter activation times should be used for highly-chargedproteins to produce larger fragments. Activation time canalso be manipulated to influence coverage in alternateregions of the protein sequence. For example, a longeractivation time can influence the production of lower-chargefragments that correspond to the N- or C-terminus of aprotein sequence.48 Spectra were collected for alternativeactivation times – including 4 ms, 7 ms, 15 ms, 50 ms,and 100 ms. The shorter activation times resulted in richerfragmentation spectra, while longer activation times pro-duced fragments corespondent to the termini of the pro-tein sequence. Figure 7 displays ETD MS/MS fragmenta-tion spectra produced using a 4 msec (A) and 100 msecactivation time (B).

The combination of information from alternative fragmentation strategies produced the most comprehensivecoverage of the protein sequence. The sequence map shownin Figure 8 illustrates the advantage of combining infor-mation garnered from multiple activation types to definethe amino acid sequence, offering amino acid resolutionfor large parts of the N and C termini of the sequence.Complementary fragmentation mechanisms also provideincreased evidence to confirm bond linkages, with thepresence of b/y or c/z-type ions for the same breakagepoint, or coverage in alternative regions of the sequence.CID produced larger fragments representing cleavage inthe middle of the sequence. Preservation of the intact stateof the molecule through whole-molecule fragmentationavoids loss of information, which is a common consequenceof protein digestion used in traditional peptide mappingcharacterization.

Figure 7: ETD MS/MS spectrum of enolase acquired at 60,000 resolving power, isolating a m/z 100 window around m/z 790, with an activation time of 4 msec. Approximately 1000 scans were averaged (A). ETD MS/MS spectrum of enolase acquired at 60,000 resolving power, isolating a m/z 100 windowaround m/z 790, with an activation time of 100 msec. Approximately, 600 scans were averaged (B). Some example sequence ions which could be labeled in the space of the figure are shown, with corresponding mass accuracies. A detailed map of coverage can be seen in Figure 8.

Conclusions• The high resolving power of Orbitrap analyzer detectionallows isotopic resolution of proteins as large as 47 kDaas well as large fragments, facilitating top-down MS/MSexperiments.

• For proteins larger than 50 kDa (for which isotopic resolution is not possible) a resolution setting of 7500 or15,000 should be used for Orbitrap analyzer detectionto improve the signal-to-noise ratio of the mass spectra.

• High-mass-accuracy detection offered by the Orbitrapanalyzer allows more precise measurement of molecularmasses of intact proteins.

• Proper desalting and desolvation are necessary for cleanerprotein signals and better signal-to-noise ratios. Propersetting for temperatures, desolvation gases and sourcevoltages are necessary for the removal of unwanted non-covalent adducts characteristic of large proteins.

• A reduced detection delay should be used for best detection of large macromolecules in LTQ Orbitrapmass spectrometers.

• The availability of multiple activation strategies in con-junction with high-performance detection characteristicsoffered by the hybrid systems allows top-down proteincharacterization with more complete sequence definition.ETD, in particular, offers amino acid resolution for largeparts of the N and C termini of the sequence.

• Reducing the gas flow from the HCD collision cell forLTQ Orbitrap XL instruments (turning the gas off)results in improved detection of large proteins in theOrbitrap analyzer.

References1. Makarov, A.; Denisov, E.; Kholomeev, A.; Balschun, W.; Lange, O.;

Strupat, K.; Horning, S. Performance Evaluation of a Hybrid Linear IonTrap/Orbitrap Mass Spectrometer. Anal. Chem. 2006, 78(7), 2113-2120.

2. Makarov, A.; Denisov, E.; Lange, O.; Horning, S. Dynamic Range of MassAccuracy in LTQ Orbitrap Hybrid Mass Spectrometer. J. Am. Soc. MassSpectrom. 2006, 17(7), 977-982.

3. McAlister, G.C.; Phanstiel, D.; Good, D.M.; Berggren, W.T.; Coon, J.J.Implementation of Electron-Transfer Dissociation on a Hybrid Linear IonTrap-Orbitrap Mass Spectrometer. Anal. Chem. 2007, 79(10), 3525-3534.

4. Olsen, J.V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M.Higher-Energy C-trap Dissociation for Peptide Modification Analysis. Nat.Methods. 2007, 4(9), 709-712.

5. McAlister, G.C.; Berggren, W.T.; Griep-Raming, J.; Horning, S.; Makarov,A.; Phanstiel, D.; Stafford, G.; Swaney, D.L.; Syka, J.E.; Zabrouskov, V.;Coon, J.J. A Proteomics Grade Electron Transfer Dissociation-EnabledHybrid Linear Ion Trap-Orbitrap Mass Spectrometer. J. Proteome Res.2008, 7(8), 3127-3136.

6. Williams D.K. Jr; McAlister G.C.; Good D.M.; Coon J.J.; Muddiman D.C. Dual electrospray ion source for electron-transfer dissociation on ahybrid linear ion trap-Orbitrap mass spectrometer. Anal. Chem. 2007, 79,7916-7919.

7. Olsen, J.V.; Blagoev, B.; Gnad, F.; Macek, B.; Kuman, C.; Mortensen, P.;Mann, M. Global, In Vivo, and Site-Specific Phosphorylation Dynamics inSignaling Networks. Cell 2006, 127, 635-648.

8. Li, X.; Gerber, S.A.; Rudner, A.D.; Beausoleil, S.A.; Haas, W.; Villen, J.; Elias,J.E.; Gygi, S.P. Large-Scale Phosphorylation Analysis of Alpha-Factor-Arrested Saccharomyces cerevisiae. J. Proteome Res. 2007, 6, 1119-1197.

9. Mann, K.; Olsen, J.V.; Macek, B.; Gnad, F.; Mann, M. Phosphoproteins ofthe Chicken Eggshell Calcified Layer. Proteomics 2007, 7, 106-115.

10. Macek, B.; Mijakovic, I.; Olsen, J.V.; Gnad, F.; Kumar, C.; Jensen, P.R.;Mann, M. The Serine/Threonine/Tyrosine Phosphoproteome of the ModelBacterium Bacillus subtilis. Mol. Cell. Proteomics 2007, 6, 607-707.

11. Wisniewski, J.R.; Zougman, A.; Kruger, S.; Mann, M. MassSpectrometric Mapping of Linker Histone H1 Variants Reveals MultipleAcetylations, Methylations, and Phosphorylation as Well as Differencesbetween Cell Culture and Tissue. Mol. Cell. Proteomics 2007, 6, 72-87.

12. Dave, K.A.; Hamilton, B.R.; Wallis, T.P.; Furness, S.G.B.; Whitelaw,M.L.; Gorman, J.J. Identification of N,N Epsilon-Dimethyl-Lysine in theMurine Dioxin Receptor Using MALDI-TOF/TOF and ESI-LTQ-Orbitrap-FT-MS. Intl. J. Mass Spectrom. 2007, 268, 168-180.

13. Villen, J.; Beausoleil, S.A.; Berber, S.A.; Gygi, S.P. Large-ScalePhosphorylation Analysis of Mouse Liver. Proc. Natl. Acad. Sci. U.S.A.2007, 104, 1488-1493.

14. Beausoleil, S.A.; Villen, J.; Gerber, S.A.; Rush, J.; Gygi, S.P. A Probability-Based Approach for High-Throughput Protein Phosphorylation Analysisand Site Localization. Nat. Biotech. 2008, 24, 1285-1292.

15. Cantin, G.T.; Yi, W.; Lu, B.; Park, S.K.; Xu, T.; Lee, J.D.; Yates, J.R., III.Combining Protein-Based IMAC, Peptide-Based IMAC, and Mudpit forEfficient Phosphoproteomic Analysis. J. Proteome Res. 2008, 7, 1346-1351.

16. Villen, J.; Gygi, S.P. The SCX/IMAC Enrichment Approach for GlobalPhosphorylation Analysis by Mass Spectrometry. Nat. Protocols 2008, 3,1630-1638.

17. Dephoure, N.; Zhou, C.; Villen, J.; Beausoleil, S.A.; Bakalarski, C.E.;Elledge, S.J.; Gygi, S.P. A Quantitative Atlas of Mitotic Phosphorylation.Proc. Natl. Acad. Sci. 2008, 105, 10762-10767.

18. Ballif, B.A.; Carey, G.R.; Sunyaev, S.R.; Gygi, S.P. Large-ScaleIdentification and Evolution Indexing of Tyrosine Phosphorylation Sitesfrom Murine Brain. J. Proteome Res 2008, 7, 311-318.

19. Wilson-Grady, J.T.; Villen, J.; Gygi, S.P. Phosphoproteome Analysis ofFission Yeast. J. Proteome Res. 2008, 7, 1088-1097.

20. Sugiyama, N.; Nakagami, H.; Mochida, K.; Daudi, A.; Tomita, M.;Shirasu, K.; Ishihama, Y. Large-Scale Phosphorylation Mapping Revealsthe Extent of Tyrosine Phosphorylation in Arabidopsis. Mol. SystemsBiol. 2008, 4, 193-199.

21. Mertins, B.; Eberl, H.C.; Renkawitz, J.; Olsen, J.V.; Tremblay, M.L.;Mann, M.; Ullrich, A.; Daub, H. Investigation of Protein-TyrosinePhosphatase 1B Function by Quantitative Proteomics. Mol. Cell.Proteomics 2008, 7, 1763-1777.

22. Phanstiel, D.; Zhang, Y.; Marto, J.A.; Coon, J.J. Peptide and ProteinQuantification Using iTRAQ with Electron Transfer Dissociation. J. Am.Soc. Mass Spectrom. 2008, 19(9), 1255-1262.

23. Zhang, Y.; Ficcaro, S.B.; Li, S.; Marto, J.A. Optimized Orbitrap HCD forQuantitative Analysis of Phosphopeptides. J. Am. Soc. Mass Spectrom.2009, 20, 1425-1434.

24. Viner, R.I.; Zhang, T.; Second, T.; Zabrouskov, V. Quantification of Post-Translationally Modified Peptides of Bovine alpha–Crystallin UsingTandem Mass Tags and Electron Transfer Dissociation. J. Proteomics2009, 72, 874-885.

Figure 8: Coverage map combining multiple activation strategies to definethe sequence of yeast enolase upon whole protein fragmentation. Xtractsoftware was used to deconvolute fragmentation spectra to a monoisotopicpeak mass list, which was then matched against the sequence with ProSightPCsoftware. Mass accuracy of all matched fragments was less than 5 ppm.Respective fragments detected with each activation type are color-codedaccording to the legend.

Part of Thermo Fisher Scientific

AN63294_E 08/10M

www.thermoscientific.comLegal Notices: ©2010 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

Thermo Fisher Scientific,San Jose, CA USA is ISO Certified.

Thermo Fisher Scientific (Bremen)GmbH Management SystemRegistered to ISO 9001:2008

In addition to these

offices, Thermo Fisher

Scientific maintains

a network of represen -

tative organizations

throughout the world.

Africa-Other+27 11 570 1840Australia+61 3 9757 4300Austria+43 1 333 50 34 0Belgium+32 53 73 42 41Canada+1 800 530 8447China+86 10 8419 3588Denmark+45 70 23 62 60 Europe-Other+43 1 333 50 34 0Finland /Norway /Sweden+46 8 556 468 00France+33 1 60 92 48 00Germany+49 6103 408 1014India+91 22 6742 9434Italy+39 02 950 591Japan +81 45 453 9100Latin America+1 561 688 8700Middle East+43 1 333 50 34 0Netherlands+31 76 579 55 55New Zealand+64 9 980 6700Russia/CIS+43 1 333 50 34 0South Africa+27 11 570 1840Spain+34 914 845 965Switzerland+41 61 716 77 00UK+44 1442 233555USA+1 800 532 4752

25. Zybailov, B.; Sun, Q.; van Wijk, K.J. Workflow for Large Scale Detectionand Validation of Peptide Modifications by RPLC-LTQ-Orbitrap:Application to the Arabidopsis Thaliana Leaf Proteome and an OnlineModified Peptide Library. Anal. Chem. 2009, 81(19), 8015-8024.

26. Wu, H.Y.; Tseng, V.S.; Chen, L.C.; Chang, Y.C.; Ping, P.; Liao, C.C.;Tsay, Y.G.; Yu, J.S.; Liao, P.C. Combining Alkaline PhosphataseTreatment and Hybrid Linear Ion Trap/Orbitrap High Mass AccuracyLiquid Chromatography-Mass Spectrometry Data for the Efficient andConfident Identification of Protein Phosphorylation. Anal. Chem. 2009,81(18), 7778-7787.

27. Boja, E.S.; Phillips, D.; French, S.A.; Harris, R.A.; Balaban, R.S.Quantitative Mitochondrial Phosphoproteomics Using iTRAQ on anLTQ-Orbitrap with High Energy Collision Dissociation. J. Proteome Res.2009, 8(10), 4665-4675.

28. Whelan, S.A.; Lu, M.; He, J.; Yan, W.; Saxton, R.E.; Faull, K.F.;Whitelegge, J.P.; Chang, H.R. Mass Spectrometry (LC-MS/MS) Site-Mapping of N-Glycosylated Membrane Proteins for Breast CancerBiomarkers. J. Proteome Res. 2009, 8(8), 4151-4160.

29. Darula, Z.; Chalkley, R.J.; Baker, P.; Burlingame, A.L.; Medzihradszky,K.F. Mass Spectrometric Analysis, Automated Identification andComplete Annotation of O-Linked Glycopeptides. Eur. J. Mass Spectrom.2010, 16(3), 421-428.

30. Segu, Z.M.; Mechref, Y. Characterizing Protein Glycosylation SitesThrough Higher-Energy C-Trap Dissociation. Rapid Commun. MassSpectrom. 2010, 24(9), 1217-1225.

31. Eliuk, S.M.; Maltby, D.; Panning, B.; Burlingame, A.L. High ResolutionElectron Transfer Dissociation Studies of Unfractionated Intact Histonesfrom Murine Embryonic Stem Cells Using On-Line Capillary LCSeparation: Determination of Abundant Histone Isoforms and Post-Translational Modifications. Mol. Cell Proteomics. 2010, 9(5), 824-837.

32. Jung, H.R.; Pasini, D.; Helin, K.; Jensen, O.N. Quantitative MassSpectrometry of Histones H3.2 and H3.3 in Suz12-Deficient MouseEmbryonic Stem Cells Reveals Distinct, Dynamic Post-TranslationalModifications at Lys-27 and Lys-36. Mol. Cell Proteomics 2010, 9(5),838-850.

33. Ge, F.; Xiao, C.L.; Yin, X.F.; Lu, C.H.; Zeng, H.L.; He, Q.Y.Phosphoproteomic Analysis of Primary Human Multiple Myeloma Cells.J. Proteomics 2010, 73(7), 1381-1390.

34. Mischerikow, N.; Altelaar, A.F.; Navarro, J.D.; Mohammed, S.; Heck, A.Comparative Assessment of Site Assignments in CID and ETD Spectra ofPhosphopeptides Discloses Limited Relocation of Phosphate Groups. MolCell Proteomics 2010, Mar 16. [E-publication ahead of print]

35. Chen, Y.; Hoehenwarter, W.; Weckwerth, W. Comparative Analysis ofPhytohormone-Responsive Phosphoproteins in Arabidopsis ThalianaUsing Tio-Phosphopeptide Enrichment and Mass Accuracy PrecursorAlignment. Plant J. 2010, Mar 31. [E-publication ahead of print]

36. Frank, A.M.; Savitski, M.M.; Nielsen, M.L.; Zubarev, R.A.; Pevzner, P.A.De Novo Peptide Sequencing and Identification with Precision MassSpectrometry. J. Proteome Res. 2007, 6(1), 114-123.

37. DiMaggio, P.A., Jr.; Floudas, C.A.; Lu, B.; Yates, J.R., III. A HybridMethod for Peptide Identification Using Integer Linear Optimization,Local Database Search, and Quadrupole Time-Of-Flight or OrbitrapTandem Mass Spectrometry. J. Proteome Res. 2008, 7(4), 1584-1593.

38. Pan, C.; Park, B.H.; McDonald, W.H.; Carey, P.A.; Banfield, J.F.;VerBerkmoes, N.C.; Hettich, R.L.; Samatova, N.F. A High-Throughput DeNovo Sequencing Approach for Shotgun Proteomics Using High-ResolutionTandem Mass Spectrometry. BMC Bioinformatics 2010, 11, 118-131.

39. Stoppacher, N.; Zeilinger, S.; Omann, M.; Lassahn, P.-G.; Roitinger, A.;Krska, R.; Schuhmacher, R. Characterization of the Peptaibiome of theBiocontrol Fungus Trichoderma Atroviride by LiquidChromatography/Tandem Mass Spectrometry. Rapid Commun. MassSpectrom. 2008, 22, 1889-1898.

40. Yates, J.R.; Cociorva, D.; Liao, L.; Zabrouskov, V. Performance of aLinear Ion Trap-Orbitrap Hybrid for Peptide Analysis. Anal. Chem.2006, 78(2), 493-500.

41. Viner, R.; Zhang, T.; Peterman, S.; Zabrouskov, V. Advantages of theLTQ Orbitrap for Protein Identification in Complex Digests. ThermoScientific Application Note 386, 2007.

42. Bantscheff, M.; Boesche, M.; Eberhard, D.; Matthieson, T.; Sweetman, G.;Kuster, B. Robust and Sensitive iTRAQ Quantification on an LTQ OrbitrapMass Spectrometer. Mol. Cell Proteomics. 2008, 7(9), 1702-1713.

43. Yates, J.R.; Ruse, C.I.; Nakorchevsky, A. Proteomics by MassSpectrometry: Approaches, Advances, and Applications. Annu. Rev.Biomed. Eng. 2009, 11, 49-79.

44. Second, T.P.; Blethrow, J.; Schwartz, J.C.; Zabrouskov, V. Novel DualPressure Linear Ion Trap Offers Breakthrough Performance in ProteomicsExperiments. Thermo Scientific Application Note 462, 2009.

45. Second, T.P.; Blethrow, J.D,; Schwartz, J.C.; Merrihew, G.E.; MacCoss,M.J.; Swaney, D.L.; Russell, J.D.; Coon, J.J.; Zabrouskov, V. Dual-Pressure Linear Ion Trap Mass Spectrometer Improving the Analysis ofComplex Protein Mixtures. Anal. Chem. 2009, 81(18), 7757-7765.

46. Olsen, J.V.; Schwartz, J.C.; Griep-Raming, J.; Nielsen, M.L.; Damoc, E.;Denisov, E.; Lange, O.; Remes, P.; Taylor, D.; Splendore, M.; Wouters,E.R.; Senko, M.; Makarov, A.; Mann, M.; Horning, S. A Dual-PressureLinear Ion Trap Orbitrap Instrument with Very High Sequencing Speed.Mol. Cell Proteomics. 2009, 8(12), 2759-2769.

47. McAlister, G.C.; Phanstiel, D.; Wenger, C.D.; Lee, M.V.; Coon, J.J.Analysis of Tandem Mass Spectra by FTMS for Improved Large-ScaleProteomics with Superior Protein Quantification. Anal. Chem. 2010,82(1), 316-322.

48. Hao, Z.; Jiang, L.; Mohtashemi, I.; Huhmer, A. N- and C-terminalSequencing of Proteins Using Top-Down Electron Transfer DissociationMass Spectrometry. Thermo Scientific Application Note 484, 2010.

49. Macek, B.; Waanders, L.F.; Olsen, J.V.; Mann, M. Top-Down ProteinSequencing and MS3 on a Hybrid Linear Quadrupole Ion Trap-OrbitrapMass Spectrometer. Mol. Cell Proteomics 2006, 5(5), 949-958.

50. Zhang, Z.; Shah, B. Characterization of Variable Regions of MonoclonalAntibodies by Top-Down Mass Spectrometry. Anal. Chem. 2007, 79(15),5723-5729.

51. Waanders, L.F.; Hanke, S.; Mann, M. Top-Down Quantitation andCharacterization of SILAC-labeled Proteins. J. Am. Soc. Mass Spectrom.2007, 18(11), 2058-2064.

52. Bondarenko, P.V.; Second, T.P.; Zabrouskov, V.; Makarov, A.A.; Zhang, Z.Mass Measurement and Top-Down HPLC/MS Analysis of Intact MonoclonalAntibodies on a Hybrid Linear Quadrupole Ion Trap-Orbitrap MassSpectrometer. J. Am. Soc. Mass Spectrom. 2009, 20(8), 1415-1424.

53. Makarov, A.; Denisov, E. Dynamics of Ions of Intact Proteins in the OrbitrapMass Analyzer. J. Am. Soc. Mass Spectrom. 2009, 20(8), 1486-1495.

54. Tsai, Y.S.; Scherl, A.; Shaw, J.L.; MacKay, C.L.; Shaffer, S.A.; Langridge-Smith, P.R.; Goodlett, D.R. Precursor Ion Independent Algorithm forTop-Down Shotgun Proteomics. J. Am. Soc. Mass Spectrom. 2009,20(11), 2154-2166.

55. Wynne, C.; Fenselau, C.; Demirev, P.A.; Edwards, N. Top-DownIdentification of Protein Biomarkers in bacteria with UnsequencedGenomes. Anal. Chem. 2009, 81(23), 9633-9642.

56. Zhang, J.; Liu, H.; Katta, V. Structural Characterization of IntactAntibodies by High-Resolution LTQ Orbitrap Mass Spectrometry. J. Mass Spectrom. 2010, 45(1), 112-120.

57. Pflieger, D.; Przybylski, C.; Gonnet, F.; Le Caer, J.P.; Lunardi, T.; Arlaud,G.J.; Daniel, R. Analysis of Human C1q by Combined Bottom-Up andTop-Down Mass Spectrometry: Detailed Mapping of Post-TranslationalModifications and Insights into the C1r/C1s Binding Sites. Mol. CellProteomics. 2010, 9, 593-610.

58. Carpenter, J.E.; Jackson, W.; de Souza, G.A.; Haarr, L.; Grose, C. Insulin-Degrading Enzyme Binds to the Nonglycosylated Precursor of Varicella-Zoster Virus gE Protein Found in the Endoplasmic Reticulum. J. Virol.2010, 84(2), 847-855.