MCP Sign the guestbook
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/mcp.M500219-MCP200 on October 18, 2005.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
M500219-MCP200v1
5/1/14    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Glossary
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patrie, S. M.
Right arrow Articles by Kelleher, N. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patrie, S. M.
Right arrow Articles by Kelleher, N. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Molecular & Cellular Proteomics 5:14-25, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Top Down Mass Spectrometry of <60-kDa Proteins from Methanosarcina acetivorans Using Quadrupole FTMS with Automated Octopole Collisionally Activated Dissociation*,S

Steven M. Patrie{ddagger}, Jonathan T. Ferguson{ddagger}, Dana E. Robinson{ddagger}, Dave Whipple{ddagger}, Michael Rother§, William W. Metcalf§ and Neil L. Kelleher{ddagger},

From the Departments of {ddagger} Chemistry and § Microbiology, University of Illinois, Urbana, Illinois 61801


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
A fragmentation geometry based upon axial acceleration of m/z-selected protein ions into a linear octopole ion trap allowed simultaneous production and external accumulation of fragment ions prior to m/z measurement in a FT mass spectrometer. Improved dynamic range resulting from this octopole collisionally activated dissociation resulted in a 2.5x increase in experimental throughput and a 2x increase in fragment ion matches to gene products identified and characterized in the top down fashion. The acceleration voltage for optimal fragmentation has a m/z and mass dependence, knowledge of which facilitated an automated platform for top down MS/MS on a quadrupole FT hybrid mass spectrometer. Controlled by improved software for data acquisition (e.g. using dynamic exclusion of previously identified species), automated octopole collisionally activated dissociation of samples fractionated using chromatofocusing and reversed-phase liquid chromatography achieved a significant increase in protein identification rate versus previous benchmarks. Also a batch analysis version of ProSight PTM facilitated probability-based identification of intact proteins obtained in a higher throughput fashion. In total, 101 unique proteins (5–59 kDa) were identified from whole cell lysates of Methanosarcina acetivorans grown anaerobically, including the characterization of several mispredicted start sites and biologically relevant mass discrepancies.

Over the last 3 years a platform consisting of two-dimensional fractionation (1), an automated (2, 3) quadrupole (Q)1 FT mass spectrometer (4), and new informatic tools (5–7) has emerged for "top down" analysis (8–11) of intact proteins with DNA-predicted sequences. Application of the nascent platform to Methanococcus jannaschii and Saccharomyces cerevisiae led to the identification of ~230 expressed gene products (<39 kDa) with 100% sequence coverage and simultaneous characterization of ~50 post-translational modifications (PTMs) (12, 13). Although yielding promising results, throughput has been limited compared with bottom up platforms based on tryptic peptides (14–19).

Poor dynamic range associated with ESI (20) of intact proteins in complex mixtures, with up to 15 observed proteins per fraction, is minimized by preselection of pseudomolecular ions with quadrupole mass filter-FT or quadrupole ion trap-FT hybrid instruments (4, 21–25). Given limitations in ion transmission of the quadrupole mass filter at 1-Da resolution, isolation windows in excess of {Delta}m/z = 5 were commonly used in prior studies for multiplexed fragmentation of several precursor proteins at once and detection of hundreds of fragment ions (4). This challenges the specificity of protein identification because the search depends largely upon the mass accuracy and the ratio n/f of the number of matching fragment ions for a protein in a database (n) relative to the number observed (f) (5).

Controlled fragmentation with infrared multiphoton dissociation (26) facilitated development of an automated platform with FTMS instrumentation (2). In this strategy, dual control over both laser intensity and duration compensates for the large range of fragmentation thresholds of intact protein ions observed in fractionated mixtures of whole cell lysates. An alternative to infrared multiphoton dissociation is sustained off-resonance irradiation collisionally activated dissociation (CAD) (27). Although amenable to automation (28), increased cell pressure required to induce ion fragmentation limits MS/MS duty cycle. Electron capture dissociation (29, 30) is possible in an automated fashion (31) but results in diverse dissociation channels that often translates to co-adding many scans to increase fragment ion S/N. Recent application of external accumulation in FT mass spectrometers (32) has led to examples of ion fragmentation in the quadrupole (25, 33, 34), hexapole, (35) and octopole storage devices (22). It has also given rise to a phenomenon described as multipole storage-assisted dissociation (MSAD) (35–37) where unwanted fragmentation occurs due to high charge density in the ion accumulation device. Attempts to control this process were recently shown for proteins as large as 116 kDa (38, 39). However, its usefulness for the identification of unknown proteins from complex mixtures is not clear.

In a previous study, we described that efficient fragmentation and accumulation of the product ions could be achieved simultaneously in the accumulation region of a Q-FT mass spectrometer instrument by reducing the axial dc potential of the multipole (4). This type of fragmentation performed in an octopole is akin to CAD in a triple quadrupole mass spectrometer (40, 41). In this study we characterized and implemented this "octopole collisionally activated dissociation" (OCAD) for MS/MS on intact proteins in an automated work flow. OCAD avoids the deleterious effects of secondary fragmentation associated with high space charge, differentiating it in an important way from MSAD. OCAD works in conjunction with new software developed to facilitate automation (3) of the MIDAS data station (42) for effective fragmentation of ions that are 5–60 kDa. Finally a new two-dimensional platform was utilized for protein separation based on pI and hydrophobicity that helped to extend the mass range for intact protein identification and characterization of 101 intact proteins from whole cell lysates of Methanosarcina acetivorans (43). This signifies a step forward for top down mass spectrometry and complements the ~290 identified using a 2D gel/bottom up approach on MeOH-grown cells reported by Li et al. (44, 45).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Protein samples from M. acetivorans (C2A), grown on MeOH (46), were produced by two-dimensional fractionation of cell lysates. Two different separation platforms were used in this study. The first was continuous elution PAGE facilitated by an acid-labile surfactant (ALS) from Waters Corp. (Milford, MA) followed by reversed-phase (RP) LC (1). The second was the ProteomeLabTM PF 2D protein fractionation system from Beckman Coulter (Fullerton, CA) with chromatofocusing as the first dimension and non-porous silica RPLC in the second (43). Cell lysates were produced by suspension of cells in either lysis buffer (1) or according to protocols outlined in the PF 2D operator manual. Suspended cells were lysed by 15–30 s of microsonication. For the ALS-PAGE/RPLC run 10–15 mg of protein (determined by Bradford assay) was used. For the PF 2D run 1–5 mg of protein (determined by Bradford assay) was used. Molecular weight ranges of the ALS-PAGE fractions were determined by SDS-PAGE.

PF 2D (or ALS-PAGE/RPLC) fractions were lyophilized, resuspended in electrospray solution (78:20:2 CH3CN:H2O:CH3COOH), and analyzed on a custom Q-FT mass spectrometer (4). Samples were presented to the instrument with the NanoMate 100 from Advion BioSciences (Ithaca, NY). Ten to 20 µl of sample was loaded into a 96-well plate and covered with an aluminum seal. This sample load reliably gave over 1 h of running time with ~200 nl/min typical flow rate at 0.2 p.s.i. back pressure and a chip voltage of 1500 V. Solvents and other reagents were obtained from Sigma.

Automation of the MIDAS data station (42) was facilitated by a custom tool command language library with THRASH (47) directed processing (3). In brief, a broadband spectrum (~10 scans) of intact pseudomolecular ions was obtained followed by sampling of a 200–300 {Delta}m/z region of the spectrum in consecutive 60 m/z windows (10–16 scans each) with the quadrupole mass filter, defined as a data-independent "quad-march" experiment (3). Each mass spectrum (for intact proteins) was processed with a remote version of the THRASH algorithm with three processing protocols: zero truncations, one zero fill, Hamming apodization from 450–2000 m/z; one truncation, one zero fill, Hamming apodization from 450–2000 m/z; and zero truncations, one zero fill, Hamming apodization from 550–1800 m/z. Only intact masses observed with at least three different charge states at S/N >3 were considered for MS/MS. All masses are monoisotopic unless presented with an italicized "-X" where "X" represents the most abundant isotope in the isotopic envelope (a "0" corresponds to the monoisotopic mass).

Software to complement this THRASH-directed approach was developed in-house. Generated lists of pseudomolecular ions from the broadband and quad-march spectra were combined and filtered with a custom data filter to obtain protein charge state distributions. The filter enables features such as dynamic exclusion of previously identified proteins and exclusion of adduct peaks (e.g. phosphate, sodium, potassium, and oxidation). The filter also integrates user definable searches of molecular ion spectra to facilitate detection of common post-translational modifications of known mass shifts. After the list of species to be targeted was created (typically four to six per sample), new tool command language scripts were generated with automatically adjusted variables such as accumulation and scan number based on the observed ion mass and intensity.

Experimental MS/MS mass peak lists were analyzed for b and y ions and processed on-the-fly with a remote version of the THRASH algorithm. MS/MS spectra were processed with three different levels of truncation: 0, 1, and 2, which corresponds to 512K, 256K, and 128K processing of the original 512K dataset. Protein identification was performed using a command line version of ProSight PTM (6, 7) used for batch processing of multiple MS/MS spectra without user intervention. Identification of the protein was based on any of the three individual datasets. The probability scores <0.01 with at least seven matching ions (the estimated minimum number of fragment ions needed to achieve 99% confidence in protein assignment in the most gene-dense region of the M. acetivorans database) were retained from the search with fragment ion tolerance set at ±30 ppm to accommodate externally calibrated ions. To accommodate mass shifts associated with post-translational modifications, proteolysis and parallel fragmentation of multiple species, the entire database was searched in 1000-Da increments.

In some cases, manual scrutiny of {Delta}m values of an identified protein was performed in the Single Protein Mode of ProSight PTM to characterize selected proteins (6). Combination of THRASH outputs for differently processed MS/MS datasets can increase the degree of localization for {Delta}m values in the protein by inclusion of all fragment ions that are optimized for either resolution (512K dataset) or S/N (128K dataset).

Ions were accumulated in an 18-cm octopole (1.5 MHz at 500 volts peak-to-peak –10-V dc offset) after traversing through the quadrupole (ABB Extrel trifilter (Pittsburgh, PA), 20-cm-long x 9.5-mm-diameter rods, controlled by a 1.2-MHz 300-watt QC150 rf power supply, –25-V offset) in either rf-only or mass selection mode. Nitrogen gas (~1 millitorr) facilitated accumulation and dissociation in this region. For improved transmission through the quadrupole, ions were collisionally axialized in a 20-cm octopole (1.5 MHz at 500 volts peak-to-peak –10-V dc offset at 1 millitorr, located behind the skimmer) for 100 ms prior to transfer through the quadrupole. The transfer was then repeated as necessary to accumulate the desired ion population. Direct pressure readings were not available for the octopole accumulation region (~low millitorr), so relative adjustments in pressure were monitored via an ionization gauge from Helix Technologies (Longmont, CO) in an adjacent region in the vacuum chamber. For characterization of OCAD fragmentation, a secondary manually controlled power supply was coupled to the octopole dc offset, supplied by MIDAS, to allow adjustments of the fragmentation power. During automation runs the OCAD voltage was automatically calculated and set by the MIDAS data station via the support software described above. Octopole rf and amplitude were controlled via a 33120A 15-MHz frequency generator from Hewlett Packard.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Robust Fragmentation during Ion Transit into Linear Octopole Ion Trap—
Dissociation of quadrupole-selected ions was induced by increasing the potential difference between the dc offsets of the focusing octopole and the accumulation octopole as depicted in Fig. 1, a and b. This OCAD procedure resulted in a pronounced decrease in parent ion and an increase in fragment ion populations in the FT broadband spectrum as shown with carbonic anhydrase (29.6 kDa) in Fig. 1c.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 1. External ion optics for the Q-FT mass spectrometer highlighting components used for OCAD. a, schematic highlighting the voltages applied between the focusing octopole and the accumulation octopole, which dictates the degree of fragmentation by OCAD. b, representative voltages placed on different lenses during the transfer of ions from the focusing octopole, through the filtering quadrupole, to the accumulation octopole (A.Oct) with (red)/without (green) OCAD. c, OCAD-induced fragmentation for the mass-selected 33+ charge state for carbonic anhydrase at various accumulation octopole voltages. 31+ and 32+ charge states are present due to charge transfer of the 33+ with neutral ions in the accumulation octopole after mass selection.

 
As shown for bovine ubiquitin (Supplemental Fig. 1), the number of product ions (and their size) varied with the potential offset of the accumulation octopole. Comparison of fragment ion abundance versus mass (Supplemental Fig. 1, b and c) showed water loss from the 11+ precursor as the dominant fragmentation product until –30 V at which point the y58 (6.5-kDa) fragment ion was the base peak. At –40 V both the y58 and y24 ion (2.7 kDa) were of approximately equal abundance, whereas at <–40 V the y24 ion dominated. The general trend shown for ubiquitin held true for most proteins where the average fragment ion size could be controlled by the axial well depth of the accumulation octopole.

To further characterize the ion optics, mass-selected 13+ ions for bovine ubiquitin, (the charge state with the fastest dissociation kinetics (48)) were stored within the accumulation region prior to reduction of the voltage on the accumulation octopole (Fig. 1b). Immediately after injection and storage, the potential on the accumulation octopole was decreased to –50 V and held for up to 40 s with no observable fragmentation for the mass selected species (data not shown). Further radio frequency and amplitude had negligible effect on the onset of fragmentation (data not shown). Our data indicate that the dominant mechanism for the octopole fragmentation can be attributed to the axial acceleration of ions into the front of the octopole where collisions with the buffer gas induce fragmentation as carried out in the CAD measurements in other types of mass spectrometers, e.g. triple quadrupoles (40, 41).

Dynamic Range Improvements for MS/MS—
OCAD performed on a M. acetivorans protein at –35 V yielded the results of Fig. 2 where the observed fragment ion abundances increased with accumulation time, but their identities did not change appreciably. This further indicates that the OCAD mechanism involves a fast loss of kinetic energy very soon after entering and fragmenting in the ~10-millitorr environment in the accumulation octopole. The improved S/N by extended accumulation of fragment ions provided increased identification confidence and protein characterization power. For the identified protein in Fig. 2, 12, 36, and 47 fragment ions matched when using 5-, 20-, and 50-s accumulation periods, respectively (Fig. 3, a–c). The number of fragment ion matches for a single 50-s scan was slightly better than that observed for 10 co-added 10-s scans (Fig. 3, c versus d). A total of 140 s was required for the 10 co-added scans; thus a ~2.5-fold improvement in experiment throughput was achieved by extended accumulation of the fragment ions. This is largely attributed to improved duty cycle with more of the experimental event sequence devoted to accumulation of the fragment ions and less associated with the transfer, excitation, and detection events.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 2. OCAD of a M. acetivorans protein. Shown is OCAD at –35 V of a mass-selected M. acetivorans protein ([M + 12H]12+) at 5-s (a), 20-s (b), 50-s (c), and 10-s (d) (10 co-added scans) accumulation.

 


View larger version (44K):
[in this window]
[in a new window]
  		FIG. 3. Comparison of OCAD data presented in Fig. 2. Shown is the primary sequence for identified M. acetivorans protein groES (MA0630) from OCAD data presented in Fig. 2 at 5-s (a), 20-s (b), 50-s (c), and 10-s (d) 10 co-added scans. Dividers indicate points of N-terminal (b ions) and C-terminal (y ions) cleavage. e, plot indicating number of fragment matches versus total accumulation time. Percent values indicate the relative ratio of number of fragment ions matched to MA0630 relative to the total number of ions detected multiplied by 100%.

 
With increased confidence based on the number of fragment matches for the assigned protein, overall P-scores dropped with an observed plateau ~1 x 10–35. This plateau was attributed to the observed n/f ratio, which did not change dramatically from the 20- to 50-s accumulation times (Fig. 3e). The plateau of n/f at ~70% can be explained by MSAD-produced secondary fragmentation from increased ion space charge resulting in unassigned fragment ions in the observed spectrum (internal ions not searched). However, upon comparison of a single 10-s scan with an experiment of 10 co-added 10-s scans, a similar decrease in fragment matches to total fragment numbers was observed. This indicates that at low accumulation times there are low abundance fragment ions not matching the predicted b/y ions for the protein, which can only be observed by increased accumulation time or co-adding scans. The lack of space charge-induced fragmentation (i.e. MSAD) is typical for standard operation of this instrument with accumulations times <30 s and protein concentrations <10 µM.

Comparing OCAD in an 18-cm octopole with fragmentation in commercial 6-cm hexapoles used in prior studies (35–37, 40), OCAD had less dependence on ion charge density and rf amplitude leading to a reduced propensity for unwanted (MSAD) fragmentation. This is due to the greater space charge capacity of the higher order multipole and its increased length. This lessens the pronounced energy exchange with the rf field that increases ion internal energies leading to dissociation via ion-neutral collisions (33).

OCAD Fragmentation for the Generation of Sequence Tags—
The protein discussed above under "Dynamic Range Improvements for MS/MS" was not identified during the standard Absolute Mass Search using ProSight PTM. The fragmentation data (Fig. 2c) was passed to the Sequence Tag Compiler in ProSight PTM, which returned with three sequence tags: KEEVTK, VIAVGT, and IYGGYQADEIEI. Using the Sequence Tag Search of ProSight PTM, the database of M. acetivorans was interrogated, and the groES protein (MA0630) was uniquely identified. A {Delta}m of –2070.3 Da was observed between the theoretical (12524.7-0 Da) and the observed (10454.4-0 Da) molecular mass. Single Protein Mode in ProSight PTM enabled localization of the {Delta}m to the N-terminal side of the protein with >95% of the observed fragment ions being b-type. This explains why despite an information-rich MS/MS spectrum containing almost exclusively b ions (e.g. Fig. 2c) no confident identification could be made in the Absolute Mass Search mode of ProSight. The most probable explanation for the N-terminal discrepancy is a mispredicted start site due to an error in the genome sequence resulting in an N-terminal {Delta}17 deletion and replacement of the N-terminal valine with methionine (Fig. 4, right). The new theoretical value matched within 10 ppm of the observed value. This agrees with a similar sequence discrepancy found in a groES protein from Mycobacterium tuberculosis (49). A similar truncation is observed in the closely related organisms Methanosarcina barkeri and Methanosarcina mazei (see the protein database). In these cases the truncated form is predicted by the published nucleotide sequences, suggesting that the sequence in such organisms is easily modified to produce the required start signal. Fig. 4 shows the functionality of ProSight PTM that led to the identification of the groES protein.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 4. Functionality of ProSight PTM that led to the identification and characterization of the groES protein (MA0630) from Fig. 3. Mass values correspond to monoisotopic values. Theor., theoretical; Exptl., experimental.

 
Automating OCAD—
With OCAD on a Q-FT mass spectrometer instrument, the axial dc offset can be adjusted to optimize fragmentation for the mass and charge of selected species (Fig. 5a). At various pressures, a linear response was observed for the OCAD voltage with respect to the m/z of a myoglobin charge state. An increased acceleration voltage was required to achieve the same extent of precursor ion reduction for lower charge states (or at higher pressures). For three standard proteins of varying mass at static pressure (Fig. 5b), the same linear response to OCAD voltage was observed over the 700–1200 m/z range. The y intercept for each of the lines was different with the mass of the intact protein having the largest (most highly charged) proteins requiring lower acceleration voltage for optimal fragmentation. Combining the equations for OCAD potential as a function of both m/z and mass allows for automatic adjustment of the acceleration potential during MS/MS (Equation 1).

(Eq. 1)



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 5. OCAD of standard proteins. a, OCAD voltages required to cause a 10x reduction in precursor ion signals for horse heart myoglobin charge states as a function of pressure. b, OCAD voltages required to cause a 10x reduction in precursor ion signals for three standard proteins at constant pressure (3.3E–5 torr measured in the chamber outside the octopole).

 
Automated Fragmentation with OCAD—
Application of OCAD with THRASH-directed automation is shown in Fig. 6. A single protein dominated one particular broadband spectrum (Fig. 6a). A quad-march of the 800–1000 m/z region of the spectrum yielded 397 pseudomolecular ions of which 223 were eliminated from the intact mass candidate list by a filtering program because they were either 1) singly charged contaminant species or 2) adducts due to oxidation, inorganic phosphate, or sodium, or 3) the intact mass only occurred once in all quad-march spectra. The remaining 174 ions were part of ~15 distinct charge state distributions. The nine most abundant species were targeted for mass selection and fragmentation with MS/MS spectra for three of the four identified proteins shown in Fig. 6, b and c. A total of 42 min was required for the entire automated experiment with ~9 min for the broadband/quad-march experiments and 33 min to isolate and fragment the nine intact protein charge states.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 6. Automated processing with OCAD. a, broadband ESI-Q-FTMS mass spectrum for one 2D M. acetivorans fraction (1.0-s accumulation, one scan). Shown are the mass selection (6-s accumulation, three scans) (b) and fragmentation (6-s accumulation, five scans) (c) of molecular ions observed in the original spectra of intact ions (broadband and quad-march).

 
For the 15.6- and 7.9-kDa proteins of Fig. 6b, a 100x and 200x increase in S/N was achieved by extended accumulation after mass selection, respectively. Such improvements in S/N led to detection and localization of {Delta}m values in three of four proteins identified (Table I). Characterization of {Delta}m values was highly dependent upon the number of fragment ions matching with site-specific localization of a deamidation and disulfides achieved.


View this table:
[in this window]
[in a new window]
  		TABLE I Partial list of proteins identified from M. acetivorans

 
Medium Throughput Top Down Mass Spectrometry with Automated OCAD—
An ALS-PAGE/RPLC run (~28 ALS-PAGE fractions) from M. acetivorans was analyzed using the THRASH-directed automation OCAD. Sixty-five proteins were identified with P-scores <0.01 (Table II). Improved dynamic range associated with OCAD yielded an average of 14 b/y fragment ions per protein compared with seven b/y fragment ions from previous studies (5, 6), greatly increasing the specificity of intact protein identification. Of all the proteins identified, 20% were from 0–10 kDa, 60% were from 10–20 kDa, 10% were from 20–30 kDa, and the rest were from above 30 kDa with several of these attributed to proteolysis events in the cell or during sample preparation (1). The majority of the proteins identified were conserved, hypothetical, ribosomal, and/or predicted proteins with a small percentage associated with ATP synthesis or involved in methanogenesis (46).


View this table:
[in this window]
[in a new window]
  		TABLE II Identified proteins from M. acetivorans using top down mass spectrometry

 
For the proteins identified, 66% of those observed matched theoretical masses to within 2 Da. Three of these had disulfide bonds indicating incomplete cysteine reduction during sample preparation. Of the remaining 34%, four had mispredicted start sites Table I), and four were truncated versions of the predicted protein with all fragment ions associated with one terminal end of the protein. Finally 12 proteins were identified with {Delta}m values >10 Da, which could not be explained with the fragmentation dataset. For six of the 12, the identification came in a multiplexed format; manual validation of intact masses could not be performed due to low S/N levels.

In a recent set of studies published in 2004, the first detailed investigation into the M. acetivorans proteome was presented yielding identification for ~10% of the predicted ORFs. Of the 412 identified proteins, 122 proteins were unique to acetate-grown versus 102 unique proteins for methanol-grown cells providing insight into differential protein expression (44, 45). A comparison of the identified proteins obtained with the ALS-PAGE/RPLC method with those identified by the complementary bottom up-based method showed a significant bias toward the identification of proteins with higher pI. Of the 37 proteins identified with the top down approach (that were not identified by the complementary method), 23 of them had a pI >9. The identification of proteins with high pI is not surprising because the separation method used does not discriminate based upon this characteristic. However, the ionization efficiency in the mass spectrometer should be greater for the more basic proteins. Also a direct comparison of the two complementary methods is difficult due to the limited coverage of this pI region in the bottom up method.

Enhanced Throughput and Mass Range with Chromatofocusing and RPLC—
For ALS-PAGE/RPLC, discrete molecular mass bands (~7 kDa) were reported for S. cerevisiae (12); however, the application of this separation platform to thermophiles from the Archaea has proven difficult with poor resolution observed from the ALS separation (13). Similarly mass spectrometric analysis of the M. acetivorans ALS-PAGE fractions was significantly biased toward lower molecular mass (<25 kDa) species because of their presence in the higher molecular weight fractions as well as reduced concentration of the large proteins in their respective fractions.

As an alternative, chromatofocusing (43) was used for the first dimension to separate proteins based on pI. Fig. 7a contains a chromatogram for a whole cell lysate from M. acetivorans. From pH 8.5 to pH 4.0, increasingly acidic proteins eluted from the column. Fig. 7, b and c, contains representative RPLC traces for fractions from Fig. 7a. Extended gradients were implemented to lower the number of species per fraction (such as Fig. 7c) for first dimension fractions with high absorbance values.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 7. Processing of M. acetivorans whole cell lysates with chromatofocusing, RPLC, and automated OCAD-Q-FTMS. a, PF 2D chromatogram for one M. acetivorans cell lysate. b, RPLC chromatogram for fractions 13–14 (pH 7.3–7.9) from the PF 2D run. c, RPLC chromatogram for fraction 2 (proteins with pI >8.5) with proteins identified utilizing automated OCAD-Q-FTMS. mAU, milliabsorbance units.

 
Automated MS/MS using OCAD of the 60 RPLC fractions, contained in Fig. 7c, identified 56 intact proteins from 5–59 kDa with an average of 14 b/y ions and P-scores <0.001. Twenty-one of the 56 proteins identified are >20 kDa. The increased mass range relative to the analogous ALS-PAGE runs is largely attributed to the high resolution observed for elution of proteins off the chromatofocusing column and reduced oxidation of proteins during 2D fractionation. An extended mass range for top down protein identification of whole cell lysates is shown in Fig. 8. MA4159, H+-transporting ATP synthase, subunit B (50.3 kDa), was identified with 20 b/y fragment matches yielding a seven-amino acid sequence tag. One-hundred percent characterization of the primary amino acid sequence was not achieved, and an apparent mass shift of +20 Da exists; although heavy adduction from sodium (+22) and phosphate (+98) limited clear assignment of the intact mass. This extent of salt adduction was not observed at lower mass, implying this large protein was not fully denatured during sample preparation. In the future, improving denaturing conditions during sample preparation should help to minimize adduction and further extend the mass range for top down proteomics. The optimization of sample pretreatment to minimize potentially artifactual modifications (e.g. proteolytic cleavage, oxidation, and deamidation) remains a challenge for the future.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 8. Identification of a M. acetivorans protein at high mass. a, 50.3-kDa protein identified with top down proteomics on a Q-FT mass spectrometer. Fragmentation yielded 12 unique ions (eight b and 12 y ions). b, the protein was identified as MA4159, a H+-transporting ATP synthase, subunit B. Theor., theoretical; Exptl., experimental.

 
Continued application of the platform is currently underway for the rest of the M. acetivorans PF 2D fractions promising to be the largest scale top down project to date. As conveyed in Table I, four proteins had mispredicted translational start sites, implying an error rate of ~2% in automated annotation of ORFs in microbial genomes. Further four disulfides were detected (Table II) with the one on MA1775 thought to be relevant in vivo due to the CXXC motifs present in these proteins. Lastly six {Delta}m values were unexplained and are under further investigation. With a more automated Q-FT mass spectrometer and improved fragmentation coupled with chromatofocusing and RPLC, an approximate analysis rate of ~100 proteins/week will be attainable in a sustained manner.

Conclusions—
Limitations associated with electrospray of proteins mixtures with up to 15 detectable proteins per sample have been significantly improved by incorporation of a quadrupole mass filter into the ion optics of a FTICR mass spectrometer. Also improved software for handling the filtering and selection of intact masses for MS/MS facilitates the automated acquisition of MS/MS datasets. Application of OCAD in an automated fashion extended the dynamic range of MS/MS for improved detection of structurally informative fragment ions. The ~2-fold increase in fragment ion matches increased both the specificity of protein identification and the degree of PTM localization for proteins interrogated in the top down manner. Furthermore a 2.5x increase in throughput was observed with OCAD due to increased duty cycle, resulting in identification of a projected ~100 proteins/week up to 60 kDa. Also improved resolution associated with the chromatofocusing-based separation extended the mass range for protein detection. Future emphasis on sample preparation to fully denature large molecules promises to enhance intact protein assignment for proteins >60 kDa. Future improvements in chromatographic resolution will continue to improve throughput for top down mass spectrometry. Finally implementation of an automatic gain feature to control ion populations (and reduce fragment ion tolerances to <2 ppm) should greatly improve specificity for protein identification.


  FOOTNOTES
 
Received, July 18, 2005, and in revised form, October 17, 2005.

Published, MCP Papers in Press, October 18, 2005, DOI 10.1074/mcp.M500219-MCP200

1 The abbreviations used are: Q, quadrupole; CAD, collisionally activated dissociation; OCAD, octopole collisionally activated dissociation; MSAD, multipole storage-assisted dissociation; 2D, two-dimensional; ALS, acid-labile surfactant; RP, reversed-phase; PTM, post-translational modification; rf, radio frequency; S/N, signal to noise ratio; dc, direct current; PF, protein fractionation. Back

* This work was supported by the Packard Foundation and National Science Foundation Grant CHE-0134953. Back

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

To whom correspondence should be addressed: Dept. of Chemistry, 53 RAL, University of Illinois, 600. S. Mathews, Urbana, IL 61801. Tel.: 217-244-3927; Fax: 217-244-8068; E-mail: kelleher{at}scs.uiuc.edu


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Meng, F., Cargile, B. J., Patrie, S. M., Johnson, J. R., McLoughlin, S. M., and Kelleher, N. L. (2002) Processing complex mixtures of intact proteins for direct analysis by mass spectrometry. Anal. Chem. 74, 2923 –2929[Medline]

  2. Johnson, J. R., Meng, F. Y., Forbes, A. J., Cargile, B. J., and Kelleher, N. L. (2002) Fourier-transform mass spectrometry for automated fragmentation and identification of 5–20 kDa proteins in mixtures. Electrophoresis 23, 3217 –3223[CrossRef][Medline]

  3. Patrie, S. M., Robinson, D. E., Meng, F., Du, Y., and Kelleher, N. L. (2003) Strategies for automating top-down protein analysis with Q-FTICR MS. Int. J. Mass Spectrom. 234, 175 –184

  4. Patrie, S. M., Charlebois, J. P., Quinn, J. P., Whipple, D., Marshall, A. G. Hendrickson, C. L., and Kelleher, N. L. (2004) Construction of a hybrid quadrupole/Fourier transform ion cyclotron resonance mass spectrometer for versatile MS/MS above 10 kDa. J. Am. Soc. Mass Spectrom. 15, 1099 –1108[CrossRef][Medline]

  5. Meng, F., Cargile, B. J., Miller, L. M., Forbes, A. J., Johnson, J. R., and Kelleher, N. L. (2001) Informatics and multiplexing of intact protein identification in bacteria and the archaea. Nat. Biotechnol. 19, 952 –957[CrossRef][Medline]

  6. Taylor, G. K., Kim, Y. B., Forbes, A. J., Meng, F. Y., McCarthy, R., and Kelleher, N. L. (2003) Web and database software for identification of intact proteins using ‘top down‘ mass spectrometry. Anal. Chem. 75, 4081 –4086[Medline]

  7. LeDuc, R. D., Taylor, G. K., Kim, Y. B., Januszyk, T. E., Bynum, L. H., Sola, J. V., Garavelli, J. S., and Kelleher, N. L. (2004) ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry. Nucleic Acids Res. 32, W340 –W345[Abstract/Free Full Text]

  8. Kelleher, N. L. (2004) Top-down proteomics. Anal. Chem. 76, 196A –203A

  9. Kelleher, N. L. (2000) From primary structure to function: biological insights from large-molecule mass spectra. Chem. Biol. 7, R37 –R45[CrossRef][Medline]

  10. Bogdanov B., and Smith, R. D. (2005) Proteomics by FTICR mass spectrometry: top down and bottom up. Mass Spectrom. Rev. 24, 168 –200[CrossRef][Medline]

  11. Bergquist, J. (2003) FTICR mass spectrometry in proteomics. Curr. Opin. Mol. Ther. 5, 310 –314[Medline]

  12. Meng, F. Y., Du, Y., Miller, L. M., Patrie, S. M., Robinson, D. E., and Kelleher, N. L. (2004) Molecular-level description of proteins from Saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics. Anal. Chem. 76, 2852 –2858[Medline]

  13. Forbes, A. J., Patrie, S. M., Taylor, G. K., Kim, Y. B., Jiang, L. H., and Kelleher, N. L. (2004) Targeted analysis and discovery of posttranslational modifications in proteins from methanogenic archaea by top-down MS. Proc. Natl. Acad. Sci. U. S. A. 101, 2678 –2683[Abstract/Free Full Text]

  14. Aebersold, R., and Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422, 198 –207[CrossRef][Medline]

  15. Aebersold, R., and Goodlett, D. R. (2001) Mass spectrometry in proteomics. Chem. Rev. 101, 269 –295[CrossRef][Medline]

  16. Lin, D., Tabb, D. L., and Yates, J. R. (2003) Large-scale protein identification using mass spectrometry. Biochim. Biophys. Acta 1646, 1 –10[Medline]

  17. McDonald, W. H., and Yates, J. R. (2002) Shotgun proteomics and biomarker discovery. Dis. Markers 18, 99 –105[Medline]

  18. Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J., and Gygi, S. P. (2003) Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J. Proteome Res. 2, 43 –50[CrossRef][Medline]

  19. Smith, R. D. (2002) Advanced mass spectrometric methods for the rapid and quantitative characterization of proteomics. Comp. Funct. Genomics 3, 143 –150[CrossRef]

  20. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1990) Electrospray ionization-principles and practice. Mass Spectrom. Rev. 9, 37 –70

  21. Hendrickson, C. L. Quinn., J. P., Emmett, M. R., and Marshall, A. G. (2001) Mass-selective external ion accumulation for Fourier transform ion cyclotron resonance mass spectrometry, in Proceedings of the 49th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, Chicago, May 27–31, 2001 (CD-ROM), American Society for Mass Spectrometry, Santa Fe, NM

  22. McFarland, M. A., Hendrickson, C. L., and Marshall, A. G. (2004) Ion ‘threshing‘: collisionally activated dissociation in an external octopole ion trap by oscillation of an axial electric potential gradient. Anal. Chem. 76, 1545 –1549[Medline]

  23. Belov, M. E., Nikolaev, E. N., Anderson, G. A., Auberry, K. J., Harkewicz, R., and Smith, R. D. (2001) Electrospray ionization-Fourier transform ion cyclotron mass spectrometry using ion preselection and external accumulation for ultrahigh sensitivity. J. Am. Soc. Mass. Spectrom. 12, 38 –48[Medline]

  24. Brock, A., Horn, D. M., Peters, E. C., Shaw, C. M., Ericson, C., Phung, Q. T., and Salomon, A. R. (2003) An automated matrix-assisted laser desorption/ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometer for ‘bottom-up‘ proteomics. Anal. Chem. 75, 3419 –3428[Medline]

  25. Syka, J. E., Marto, J. A., Bai, D. L., Horning, S., Senko, M. W., Schwartz, J. C., Ueberheide, B., Garcia, B., Busby, S., Muratore, T., Shabanowitz, J., and Hunt D. F. (2004) Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications. J. Proteome Res. 3, 621 –626[CrossRef][Medline]

  26. Little, D. P., Speir, J. P., Senko, M. W., O’Connor, P. B., and McLafferty, F. W. (1994) Infrared multiphoton dissociation of large multiply-charged ions for biomolecule sequencing. Anal. Chem. 66, 2809 –2815[Medline]

  27. Senko, M. W., Speir, J. P., and McLafferty, F. W. (1994) Collisional activation of large multiply-charged ions using Fourier-transform mass spectrometry. Anal. Chem. 66, 2801 –2808[Medline]

  28. Mirgorodskaya, E., O’Connor, P. B., and Costello, C. E. (2002) A general method for precalculation of parameters for sustained off resonance irradiation/collision-induced dissociation. J. Am. Soc. Mass Spectrom. 13, 318 –324[Medline]

  29. Zubarev, R. A., Kelleher, N. L., and McLafferty, F. W. (1998) Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 120, 3265 –3266[CrossRef]

  30. Zubarev, R. A., Kruger, N. A., Fridriksson, E. K., Lewis, M. A., Horn, D. M., Carpenter, B. K., and McLafferty, F. W. (1999) Electron capture dissociation of gaseous multiply-charged proteins is favored at disulfide bonds and other sites of high hydrogen atom affinity. J. Am. Chem. Soc. 121, 2857 –2862[CrossRef]

  31. Roth, M. J., Forbes, A. J., Boyne, M.T., II, Kim, Y. B., Robinson, D. E., and Kelleher, N. L. (2005) Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry. Mol. Cell. Proteomics 4, 1002 –1008[Abstract/Free Full Text]

  32. Senko, M. W., Hendrickson, C. L., Emmett, M. R., Shi, S. D. H., and Marshall, A. G. (1997) External accumulation of ions for enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. J. Am. Soc. Mass. Spectrom. 8, 970 –976[CrossRef]

  33. Belov, M. E., Gorshkov, M. V., Udseth, H. R., and Smith, R. D. (2001) Controlled ion fragmentation in a 2-D quadrupole ion trap for external ion accumulation in ESI FTICR mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 1312 –1319[Medline]

  34. Akashi, S., Takio, K., Matsui, H., Tate, S., and Kainosho, M. (1998) Collision-induced dissociation spectra obtained by Fourier transform ion cyclotron resonance mass spectrometry using a 13C, 15N-doubly depleted protein. Anal. Chem. 70, 3333 –3336[Medline]

  35. Sannes-Lowery, K., Griffey, R. H., Kruppa, G. H., Speir, J. P., and Hofstadler, S. A. (1998) Multipole storage assisted dissociation, a novel in-source dissociation technique for electrospray ionization generated ions. Rapid Commun. Mass Spectrom. 12, 1957 –1961[Medline]

  36. Hakansson, K., Axelsson, J., Palmblad, M., and Hakansson, P. (2000) Mechanistic studies of multipole storage assisted dissociation. J. Am. Soc. Mass Spectrom. 11, 210 –217[CrossRef][Medline]

  37. Sannes-Lowery, K. A., and Hofstadler, S. A. (2000) Characterization of multipole storage assisted dissociation: implications for electrospray ionization mass spectrometry characterization of biomolecules. J. Am. Soc. Mass Spectrom. 11, 1 –9[Medline]

  38. Keller, K. M., Brodbelt, J. S., Hettich, R. L., and Van Berkel, G. J. (2004) Comparison of sustained off-resonance irradiation collisionally activated dissociation and multipole storage-assisted dissociation for top-down protein analysis. J. Mass Spectrom. 39, 402 –411[Medline]

  39. Pan, C., and Hettich, R. (2005) Multipole-storage-assisted dissociation for the characterization of large proteins and simple protein mixtures by ESI-FTICR-MS. Anal. Chem. 77, 3072 –3082[Medline]

  40. Hunt, D. F., Yates, J. R., Shabanowitz, J., Winston, S., and Hauer, C. R. (1986) Protein sequencing by tandem-mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 83, 6233 –6237[Abstract/Free Full Text]

  41. Haller, I., Miraz, U. A., and Chait, B. T. (1996) Collision induced decomposition of peptides. Choice of collision parameters. J. Am. Soc. Mass Spectrom. 7, 677 –681[CrossRef]

  42. Senko, M. W., Canterbury, J. D., Guan, S. H., and Marshall, A. G. (1996) A high-performance modular data system for Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom. 10, 1839 –1844[CrossRef][Medline]

  43. Chong, B. E., Yan, F., Lubman, D. M., and Miller, F. R. (2001) Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method. Rapid Commun. Mass Spectrom. 15, 291 –296[CrossRef][Medline]

  44. Li, Q., Li, L., Rejtar, T., Karger, B. L., and Ferry, J. G. (2005) Proteome of Methanosarcina acetivorans part I: an expanded view of the biology of the cell. J. Proteome Res. 4, 112 –128[CrossRef][Medline]

  45. Li, Q., Li, L., Rejtar, T., Karger, B. L., and Ferry, J. G. (2005) Proteome of Methanosarcina acetivorans part II: comparison of protein levels in acetate- and methanol-grown cells. J. Proteome. Res. 4, 129 –135[CrossRef][Medline]

  46. Galagan, J. E., Nusbaum, C., Roy, A., Endrizzi, M. G., Macdonald, P., FitzHugh, W., Calvo, S., Engels, R., Smirnov, S., Atnoor, D., Brown, A., Allen, N., Naylor, J., Stange-Thomann, N., DeArellano, K., Johnson, R., Linton, L., McEwan, P., McKernan, K., Talamas, J., Tirrell, A., Ye, W., Zimmer, A., Barber, R. D., Cann, I., Graham, D. E., Grahame, D. A., Guss, A. M., Hedderich, R., Ingram-Smith, C., Kuettner, H. C., Krzycki, J. A., Leigh, J. A., Li, W., Liu, J., Mukhopadhyay, B., Reeve, J. N., Smith, K., Springer, T. A., Umayam, L. A., White, O., White, R. H., Conway de Macario, E., Ferry, J. G., Jarrell, K. F., Jing, H., Macario, A. J., Paulsen, I., Pritchett, M., Sowers, K. R., Swanson, R. V., Zinder, S. H., Lander, E., Metcalf, W. W., and Birren, B. (2002) The genome of M. acetivorans reveals extensive metabolic and physiological diversity. Genome Res. 12, 532 –542[Abstract/Free Full Text]

  47. Horn, D. M., Zubarev, R. A., and McLafferty, F. W. (2000) Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules. J. Am. Soc. Mass Spectrom. 11, 320 –332[CrossRef][Medline]

  48. Jockusch, R. A., Schnier, P. D., Price, W. D., Strittmatter, E. F., Demirev, P. A., and Williams, E. R. (1997) Effects of charge state on fragmentation pathways, dynamics, and activation energies of ubiquitin ions measured by blackbody infrared radiative dissociation. Anal. Chem. 69, 1119 –1126[Medline]

  49. Ge, Y., El-Naggar, M., Sze, S. K., Oh, H. B., Begley, T. P., McLafferty, F. W., Boshoff, H., and Barry, C. E. (2003) Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry. J. Am. Soc. Mass Spectrom. 14, 253 –261[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
M500219-MCP200v1
5/1/14    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Glossary
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patrie, S. M.
Right arrow Articles by Kelleher, N. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patrie, S. M.
Right arrow Articles by Kelleher, N. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?