Top-down Protein Sequencing and MS3 on a Hybrid Linear Quadrupole Ion Trap-Orbitrap Mass Spectrometer

Top-down proteomics, the analysis of intact proteins (instead of first digesting them to peptides), has the potential to become a powerful tool for mass spectrometric protein characterization. Requirements for extremely high mass resolution, accuracy, and ability to efficiently fragment large ions have often limited top-down analyses to custom built FT-ICR mass analyzers. Here we explore the hybrid linear ion trap (LTQ)-Orbitrap, a novel, high performance, and compact mass spectrometric analyzer, for top-down proteomics. Protein standards from 10 to 25 kDa were electrosprayed into the instrument using a nanoelectrospray chip. Resolving power of 60,000 was ample for isotope resolution of all protein charge states. We achieved absolute mass accuracies for intact proteins between 0.92 and 2.8 ppm using the “lock mass” mode of operation. Fifty femtomole of cytochrome c applied to the chip resulted in spectra with excellent signal-to-noise ratio and only low attomole sample consumption. Different protein charge states were dissociated in the LTQ, and the sensitivity of the orbitrap allowed routine, high resolution, and high mass accuracy fragment detection. This resulted in unambiguous charge state determination of fragment ions and identification of unmodified and modified proteins by database searching. Protein fragments were further isolated and fragmented in the LTQ followed by analysis of MS3 fragments in the orbitrap, localizing modifications to part of the sequence and helping to identify the protein with these small peptide-like fragments. Given the ready availability and ease of operation of the LTQ-Orbitrap, it may have significant impact on top-down proteomics. Top-down proteomics, the analysis of intact proteins (instead of first digesting them to peptides), has the potential to become a powerful tool for mass spectrometric protein characterization. Requirements for extremely high mass resolution, accuracy, and ability to efficiently fragment large ions have often limited top-down analyses to custom built FT-ICR mass analyzers. Here we explore the hybrid linear ion trap (LTQ)-Orbitrap, a novel, high performance, and compact mass spectrometric analyzer, for top-down proteomics. Protein standards from 10 to 25 kDa were electrosprayed into the instrument using a nanoelectrospray chip. Resolving power of 60,000 was ample for isotope resolution of all protein charge states. We achieved absolute mass accuracies for intact proteins between 0.92 and 2.8 ppm using the “lock mass” mode of operation. Fifty femtomole of cytochrome c applied to the chip resulted in spectra with excellent signal-to-noise ratio and only low attomole sample consumption. Different protein charge states were dissociated in the LTQ, and the sensitivity of the orbitrap allowed routine, high resolution, and high mass accuracy fragment detection. This resulted in unambiguous charge state determination of fragment ions and identification of unmodified and modified proteins by database searching. Protein fragments were further isolated and fragmented in the LTQ followed by analysis of MS3 fragments in the orbitrap, localizing modifications to part of the sequence and helping to identify the protein with these small peptide-like fragments. Given the ready availability and ease of operation of the LTQ-Orbitrap, it may have significant impact on top-down proteomics. Major goals in every mass spectrometry-based proteomic experiment are protein identification and characterization. Almost invariably, proteins are enzymatically degraded to peptides, which are much more amenable to mass spectrometric investigation (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5639) Google Scholar). Further advantages of this “bottom-up proteomics” approach are that one protein generates many peptides, providing many opportunities to identify or quantify it. However, identified peptides rarely cover the whole sequence of a given protein often leading to difficulties in protein characterization, particularly in determination of posttranslational modifications (PTMs). 1The abbreviations used are: PTM, posttranslational modification; LTQ, linear quadrupole ion trap; AGC, automatic gain control; PCM, polycyclodimethylsiloxane; ECD; electron capture dissociation; S/N, signal-to-noise ratio. In the alternative approach, termed “top-down proteomics,” intact proteins are ionized, physically fragmented, and analyzed in the mass spectrometer (for reviews, see Refs. 2Reid G.E. McLuckey S.A. 'Top down’ protein characterization via tandem mass spectrometry.J. Mass Spectrom. 2002; 37: 663-675Crossref PubMed Scopus (250) Google Scholar, 3Kelleher N.L. Top-down proteomics.Anal. Chem. 2004; 76: 197A-203ACrossref PubMed Google Scholar, 4Bogdanov B. Smith R.D. Proteomics by FTICR mass spectrometry: top down and bottom up.Mass Spectrom. Rev. 2005; 24: 168-200Crossref PubMed Scopus (383) Google Scholar). Because this approach starts from MS detection of the intact, fully modified protein, it has the potential for full protein characterization. Although analysis of intact proteins has been reported for almost all mass analyzers, to date only one, the FT-ICR analyzer, has sufficient resolving power and mass accuracy to efficiently analyze large protein ions. In addition, several methods especially useful for fragmentation of whole proteins have been developed for the FT-ICR analyzers, such as infrared multiphoton dissociation, sustained off-resonance irradiation, and, in particular, electron capture dissociation (ECD), which is nonergodic in nature and in some cases can cleave almost any peptide bond in proteins of up to 50 kDa (5Ge Y. Lawhorn B.G. ElNaggar M. Strauss E. Park J.H. Begley T.P. McLafferty F.W. Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry.J. Am. Chem. Soc. 2002; 124: 672-678Crossref PubMed Scopus (340) Google Scholar). A similar fragmentation method, electron transfer dissociation, was recently introduced for ion traps and, in combination with charge state reduction, shows great promise for top-down proteomics on these mass analyzers (6Coon J.J. Ueberheide B. Syka J.E. Dryhurst D.D. Ausio J. Shabanowitz J. Hunt D.F. Protein identification using sequential ion/ion reactions and tandem mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9463-9468Crossref PubMed Scopus (341) Google Scholar). CID is known to efficiently fragment proteins in ion traps, but these mass spectrometers lack sufficient resolution to resolve large protein fragment ions and their charge states. With the hybrid ion trap-FTICR mass spectrometer (LTQ-FT, Thermo Electron Corp., Bremen, Germany) it is possible to fragment large peptides or even protein ions in the ion trap and detect them with high resolution and accuracy by FT-ICR (7Wu S.L. Jardine I. Hancock W.S. Karger B.L. A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer.Rapid Commun. Mass Spectrom. 2004; 18: 2201-2207Crossref PubMed Scopus (38) Google Scholar, 8Zabrouskov V. Senko M.W. Du Y. Leduc R.D. Kelleher N.L. New and automated MSn approaches for top-down identification of modified proteins.J. Am. Soc. Mass Spectrom. 2005; 16: 2027-2038Crossref PubMed Scopus (63) Google Scholar). However, in our experience, and as shown here, the LTQ-FT is less suitable for detection of fragments produced in the LTQ due to lower sensitivity and time-of-flight effects. On the other hand, the ions can be fragmented in the ICR cell using methods like ECD and infrared multiphoton dissociation. Furthermore although the LTQ-FT is a commercial and robust instrument, the necessity for a high magnetic field detector and relatively high maintenance costs tend to limit its use to specialized laboratories. Very recently a new hybrid mass spectrometer, the LTQ-Orbitrap (Thermo Electron), was introduced (9Makarov A. Denisov E. Lange O. Kholomeev A. S. of mass accuracy in of the of the for Mass for Mass Scholar). of a linear quadrupole ion trap to a mass analyzer, the orbitrap, by A. A. a of mass Chem. PubMed Scopus Google Scholar, M. the orbitrap mass to an ion Chem. 2003; PubMed Scopus Google Scholar, A. M. a new mass Mass Spectrom. 2005; PubMed Scopus Google Scholar). In the orbitrap, ion between and their is as in the FT-ICR instrument, by their followed by of the to the mass the orbitrap is compact and magnetic field or LTQ and orbitrap are via the an which can be used to ions of known ions are and analyzed with this “lock mass accuracy for peptides is B. R. A. Lange O. S. Mann M. mass accuracy on an orbitrap mass spectrometer via into a 2005; PubMed Scopus Google Scholar). analysis with intact proteins and their fragments in the LTQ-Orbitrap has been reported In this we the of the LTQ-Orbitrap mass spectrometer for top-down analysis of proteins in mass from 10 to 25 that the instrument is of high sensitivity to femtomole high mass accuracy and isotope resolution of small In addition, protein ions can be fragmented in and in the linear ion and their fragments can be and in the We ready identification of modified and unmodified proteins by and MS3 protein standards were from proteins were cytochrome c A and and human were in with from of whole protein to of MSn A sample of was to the mass spectrometer using a A low chip was used as nanoelectrospray providing a of of were applied to the chip the power the mass spectrometer was to were using a of were on an LTQ-Orbitrap mass spectrometer (Thermo in the ion Mass accuracy was to the CID of protein charge states was in the LTQ, and fragments were and in the protein charge states the were isolated with a of and for using and an of instrument was using and the spectra were using orbitrap automated gain were to for full and for MSn Protein mass spectra were a resolving power of and MSn spectra were or mass was in all and ions and were used for as B. R. A. Lange O. S. Mann M. mass accuracy on an orbitrap mass spectrometer via into a 2005; PubMed Scopus Google Scholar). all orbitrap of 10 Protein were by using the (Thermo or by from the and charge states. protein fragment were using in by Protein of mass in protein identification MS or and database Chem. PubMed Scopus Google or for S. A. tool for proteins and Sci. PubMed Scopus Google Scholar). spectra were with the R. Kelleher N.L. and database for identification of intact proteins using mass Chem. 2003; PubMed Scopus Google the human or a custom database in A of up to was used for the protein mass to for between and due to protein ion mass was in all cases to and fragments were for protein MS3 spectra were the for protein database 2005; using the protein identification by sequence using mass PubMed Scopus Google Scholar). were as mass and fragment mass Because MS3 we the mass of to all ions that MSn spectra but in protein This ions or fragments into peptide MS3 fragments by the was a were allowed to only with were on an LTQ-FT mass spectrometer (Thermo in the ion cytochrome and were for and to the mass spectrometer using the in a as as possible to the LTQ-Orbitrap instrument was fully to all to the CID of protein charge states was in the LTQ, and fragments were and in the ICR of was used for protein charge which were for using and an of instrument was using and the spectra were using were to for full and for the MSn A resolving power of was used in of protein was used for MSn of 10 of the LTQ-Orbitrap mass spectrometer for top-down analysis of proteins was high accuracy MS of the whole protein in the orbitrap, fragmentation of a charge state of the protein of in the LTQ with detection of fragments in the orbitrap, and MS3 of CID in the LTQ and their detection in the we to for protein on the M. Mann M. of the nanoelectrospray ion Chem. PubMed Scopus Google Scholar, M. A. S. Mann M. of proteins from by mass PubMed Scopus Google is used in top-down proteomics it investigation of for of and it is Here we used a developed low nanoelectrospray chip which in the nanoelectrospray of This allowed to use a of and for more more sufficient for all We for of whole protein spectra in the orbitrap mass S/N, and accuracy were of 10 In this of 10 are to a on which is was between 10 and the resolution it was with the for the was between and to the of In of up to are possible in the LTQ part of the instrument, much the limit of of the However, in for a charge state for dissociation, only ions can be In this the ability to the be useful as B. R. A. Lange O. S. Mann M. mass accuracy on an orbitrap mass spectrometer via into a 2005; PubMed Scopus Google Scholar). the intact protein from detection of charge states were in the orbitrap for all proteins of and protein of less analyzed in this was 50 of cytochrome c that this was the protein used for and that high protein mass spectra were even one MS on a of ions attomole and with an of of cytochrome c was on the LTQ-FT instrument the with lower and and accuracies of intact proteins in the LTQ-Orbitrap and mass and of proteins used for in a new sensitivity the whole protein have been reported on FT-ICR Kelleher N.L. McLafferty F.W. protein characterization by PubMed Scopus Google have reported detection of proteins using with FT-ICR mass and Smith R.D. transform ion cyclotron resonance mass of Chem. PubMed Scopus Google have reported low detection of proteins in the mass However, these high were achieved and and or instrument nanoelectrospray in this that the orbitrap sensitivity for proteins with other used in top-down proteomics. orbitrap protein spectra were 60,000 resolution which is the resolving power of the orbitrap mass This resolution the possible of to a resolution of Although this is less the possible with high magnetic field FT-ICR analyzers, it was sufficient for isotope resolution of all proteins and and a Furthermore resolution in the orbitrap is to the of the to the as it is in the FT-ICR analyzers A. a of mass Chem. PubMed Scopus Google in of resolution the mass In this that resolution power of 60,000 of the orbitrap resolution power of of the This is useful in analysis of intact protein mass spectra protein charge states are often has to be that in the LTQ-FT instrument to resolution to up to in the orbitrap mass the is limited to and its resolving power is limited to resolution the detection of modifications such as or of small the ability to resolve of protein charge states the mass is for charge state in spectra by protein charge high mass accuracy, orbitrap spectra in this were in the mass mode of operation using ions from the as as recently B. R. A. Lange O. S. Mann M. mass accuracy on an orbitrap mass spectrometer via into a 2005; PubMed Scopus Google Scholar). In the mass orbitrap is by an of a of ions into the ions are into the and with the ion into the ion is on the and of all ions are in the intact protein the ion was used as the for the MSn the ion was used due to its in the MSn This mass accuracy up to and for protein were from up to 10 of in charge states and larger the be the isotope state was using the by and for mass characterization of large Chem. Scopus Google with the of the protein mass was from the protein spectra by all protein standards were with high mass accuracy, between 0.92 ppm and ppm absolute mass accuracy was ppm with of mass accuracy of the cytochrome c in the was ppm which was lower that in the orbitrap, due to relatively low Although FT-ICR MS is of extremely high mass are only a accuracy was 10 ppm the protein for this is the which on the ion in the ICR Although several have been to this mass resolving power for transform ion cyclotron resonance mass Commun. Mass Spectrom. Scopus Google Scholar, J.E. Smith R.D. more transform ion cyclotron resonance mass standards using Am. Soc. Mass Spectrom. PubMed Scopus Google the were the ion was or was S. R. Smith R.D. mass spectrometric analysis of intact proteins of the large using Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google have used a to as and to its In a high analysis of intact proteins of the large have identified a proteins with absolute accuracy of ppm and of ppm in cases protein mass be S. R. Smith R.D. mass spectrometric analysis of intact proteins of the large using Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). Although a of in the orbitrap mass has been we have mass as a of ion up to the limit by the of in our mass accuracies for all proteins were ppm even mass In addition, protein were with extremely high almost ppm these that the LTQ-Orbitrap can extremely high mass accuracy the protein any or of the of the top-down approach in proteomics is comprehensive characterization of of the proteins analyzed in this and A and are known to be In the of have been and the mass in the orbitrap to the mass of the protein all In A and of were in several A was as unmodified and modified with one was as unmodified and modified with one, and of these modifications were in with in a MS McLuckey S.A. identification via ion trap tandem mass of whole protein and peptide A Chem. 2003; PubMed Scopus Google Scholar). that mass from modifications were with high or significant of modifications with the such as and the intact protein In addition, it is from that sensitivity for detection of was excellent in this of protein been and the and up less of this protein mass be of use in the analysis of proteins or protein fragmentation methods have been applied to intact proteins their isolated charge to on protein In the LTQ-Orbitrap fragmentation be of the orbitrap mass fragmentation are can be fragmented by dissociation or CID in the LTQ with detection of fragment ions in the ion trap or in the using fragmentation resulted in fragmentation useful sequence all fragmentation was by CID in the reported in on the instrument, intact proteins fragment in the LTQ the used for peptide (7Wu S.L. Jardine I. Hancock W.S. Karger B.L. A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer.Rapid Commun. Mass Spectrom. 2004; 18: 2201-2207Crossref PubMed Scopus (38) Google Scholar, 8Zabrouskov V. Senko M.W. Du Y. Leduc R.D. Kelleher N.L. New and automated MSn approaches for top-down identification of modified proteins.J. Am. Soc. Mass Spectrom. 2005; 16: 2027-2038Crossref PubMed Scopus (63) Google Scholar). charge states of all analyzed proteins produced fragment ions that were and excellent and in the of intact protein spectra were as the of 10 transfer from the LTQ to the and orbitrap and of time-of-flight that fragments be in a mass fragments were by Protein R. Kelleher N.L. and database for identification of intact proteins using mass Chem. 2003; PubMed Scopus Google Scholar). Because only and fragments were using Protein of mass in protein identification MS or and database Chem. PubMed Scopus Google or S. A. tool for proteins and Sci. PubMed Scopus Google Scholar). for and fragments were with absolute accuracy ppm in the orbitrap dissociation of fragmentation of the charge state of analyzed in the orbitrap shows fragmentation that all to of CID was isotope of small and large fragments as as the mass the of one and to resolution sequence of with by and fragmentation CID of intact proteins has been in ion trap fragment ions (for a see 2Reid G.E. McLuckey S.A. 'Top down’ protein characterization via tandem mass spectrometry.J. Mass Spectrom. 2002; 37: 663-675Crossref PubMed Scopus (250) Google Scholar). this has due to the of ion traps to resolve charge states of fragment ions and necessity to charge state by of into the mass combination of a linear ion trap and the high resolution orbitrap of protein fragment spectra shows an of and CID fragmentation of its charge state in the LTQ and detection of fragment ions in the fragmentation of proteins in our were in with the reported for ion trap CID of intact proteins (7Wu S.L. Jardine I. Hancock W.S. Karger B.L. A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer.Rapid Commun. Mass Spectrom. 2004; 18: 2201-2207Crossref PubMed Scopus (38) Google Scholar, 8Zabrouskov V. Senko M.W. Du Y. Leduc R.D. Kelleher N.L. New and automated MSn approaches for top-down identification of modified proteins.J. Am. Soc. Mass Spectrom. 2005; 16: 2027-2038Crossref PubMed Scopus (63) Google Scholar, G.E. McLuckey S.A. mass of A and analysis of whole protein Chem. 2002; PubMed Scopus Google Scholar). to in and as as to were the fragmentation although other were as and In spectra of cytochrome and fragments were However, fragmented to from the and 25 from the the of the all by CID of its charge state that the all with low unmodified this all to between and of the Because the of from the peptide in the approach, was CID of intact modified fragments be fragmented further to into the of protein is known that CID fragmentation of whole proteins on the protein as the of and and that larger proteins kDa) tend to fragment in is an and a of CID that it fragmentation into a This the sensitivity for these fragments but often characterization with ECD can cleave almost all peptide in a protein Y. McLafferty F.W. Top-down mass of a protein for characterization of any posttranslational to one Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google it from relatively low which in lower sensitivity and these methods are Electron transfer dissociation, a recently developed fragmentation for ion traps, be suitable for the LTQ-Orbitrap it the advantages of nonergodic fragmentation sequence of with the high resolution, and accuracy of the orbitrap mass accuracy and of the charge states of fragment ions with the protein mass were used for protein of the fragments were to Mass the only for top-down proteomics. spectra of human were the human all other proteins were a custom database and the human protein mass was to to for potential and a of fragments with accuracy of ppm or were for a to unambiguous identification of all analyzed In the analysis of the protein was identified with 10 fragment ions absolute mass accuracy, 0.92 and of its mass was the to the of the sequence and its In the of cytochrome the mass from the mass by which to the mass of the This only shows the potential of the top-down approach to identify proteins but to to modifications in the database as A and and were identified as high their were the custom database only the human database spectra of these proteins to any This that high accuracy spectra of whole proteins in the orbitrap can be used for high database in top-down proteomics, leading to high identification and the nature of a possible of CID fragments of and was between the orbitrap and the FT-ICR mass analyzers. the fragment ions produced in the LTQ and to the ICR cell resulted in spectra of lower This resulted in a lower of identified CID fragments as with the orbitrap and sensitivity of the orbitrap mass in the MSn mode low and high mass of the FT-ICR mass spectra were particularly due to the time-of-flight the ions in the between the LTQ and the ICR However, and MSn the of the FT-ICR spectra We have shown recently that an of peptide fragmentation is on the high sensitivity and Mann M. peptide identification in proteomics by of mass spectrometric Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). with a new these of peptide With this and the excellent of the we to explore the of the LTQ-Orbitrap for top-down MS3 shows the of ion is by This ion was isolated and dissociated in the LTQ, and fragments were analyzed in the orbitrap a to a which was further by spectra many and ions to the MS3 fragment ion from the of the sequence by the see Mann M. peptide identification in proteomics by of mass spectrometric Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). MS3 spectra similar in to one of the ions of the and a less several MS3 spectra can be of a protein a into the We of shows an analyzed in the LTQ and that spectra can be of and V. Senko M.W. Du Y. Leduc R.D. Kelleher N.L. New and automated MSn approaches for top-down identification of modified proteins.J. Am. Soc. Mass Spectrom. 2005; 16: 2027-2038Crossref PubMed Scopus (63) Google on an instrument, used MS3 of proteins for of protein and protein may be sufficient to identify a protein in cases protein to fragmentation of or more proteins as in or CID of proteins in a low of leading to low in database In these unambiguous protein identification may an of as MS3 to or of in we to peptide-like fragments of proteins be fragmented and used for protein identification in a similar to protein identification in the of such a be that small fragments are less to be modified and that are relatively possible fragmentation We a small fragment of that was to be on its isotope This fragment was in the LTQ, fragmented, and analyzed in the shows a relatively MS3 similar to the of a small peptide for the were to the to be to these Because the MS3 be a or ion of the protein, were a ion to any the mass of was to the mass to it from a ion to a peptide was to only or ions are the MS3 fragments from ion Mann M. peptide identification in proteomics by of mass spectrometric Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google the peptide a by in the database only significant top was in the of the A of the database that identified the of the significant on a protein and a peptide sequence for fragments the high mass accuracy but was to the of that protein sequence and was Because the protein was modified with this shows an of modified Here we a compact LTQ-Orbitrap mass spectrometer was suitable for top-down proteomics of small We the LTQ-Orbitrap to a low nanoelectrospray providing more for a of of intact proteins sufficient to high and MS3 spectra with mass accuracies in the ppm database with the allowed identification only of unmodified but of modified Because the of proteins to that from the database this be for top-down proteomics. In we that small peptide-like fragments by protein can be fragmented to peptides in the approach, advantages of In the it be to use this instrument with on-line protein as as to new to the of the that it This may routine, high accuracy, and high analysis of the and small protein of the