| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Molecular & Cellular Proteomics 4:2010-2021, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,¶,,**
From the Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany, ¶ Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101300, China, and || Thermo Electron (Bremen) GmbH, Hanna-Kunath-Strasse 11, 28199 Bremen, Germany
 |
ABSTRACT |
|---|
 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSREFERENCES |
|---|
In a typical proteomics experiment, protein mixtures are digested to peptide mixtures that are separated by reversed phase HPLC and analyzed on-line by MS and MS/MS (6). The mass accuracy achieved in the instrument directly translates into the mass tolerances that can be specified in subsequent database searches of tandem mass spectra. Unambiguous protein identification in large data sets is by no means trivial (7), and any increase in achieved mass accuracy greatly aids the specificity of database searches in two ways (8, 9): High precursor mass accuracy in the MS spectra directly translates into fewer "candidate sequences" that need to be considered as possible matches. High mass accuracy in the MS/MS spectra leads to fewer measured fragment masses that match the calculated fragments of a candidate sequence by chance and therefore decreases the scores of false positives in database search algorithms.
In 1923, Kingdon (10) devised a method to capture ions by causing them to orbit around a central electrode. Since then, the physics community has used "Kingdon traps" in a variety of experiments, but it was always used as a capturing device, not as a mass spectrometer. A few years ago Makarov invented a novel mass analyzer, which uses orbital trapping, termed the orbitrap (11–13). On the basis of this development, a new hybrid mass spectrometer was very recently introduced commercially. It consists of the linear ion trap (LTQ) mentioned above coupled to a radio frequency (RF)-only "C-trap" for intermediate storage of ions and of the orbitrap mass analyzer (LTQ Orbitrap) (14).
In contrast to the Penning traps used in FT-ICR, the orbitrap consists of two concentric electrodes around which injected ions circle. No magnetic fields are involved, and the size of the device is only a few centimeters. The detection principle is an image current of the axial motion of ion packages orbiting the inner electrode, and the mass spectrum is obtained as the Fourier transform of this current. As a result, the device has high resolution, and it should be capable of high mass accuracy. However, mass accuracy depends on the stability of an electric field, which is more difficult to achieve than stability of a magnetic field. No detailed studies of the mass accuracy of an orbitrap for complex proteomic samples have been reported so far.
Here we show that very high mass accuracy is possible on the LTQ Orbitrap. We use a known background ion produced by electrospray in ambient conditions and inject a defined number of this ion species into the C-trap, the RF-only trap connecting the LTQ to the orbitrap. The ions constituting the mass spectrum are then added to this "lock mass," and all are injected into the orbitrap together. We furthermore average the mass values over the chromatographic elution profile of every peptide peak. Together, these steps allow average absolute mass determination in the sub-ppm range for peptides yielding at least moderate signals. The masses of peptides close to the detection limit are still determined to within a few ppm.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSREFERENCES |
|---|
SILAC Labeling of Yeast-K_out-strain—
The Saccharomyces cerevisiae strain Y15969 (BY4742; MAT 
; his3D1; leu2D0; lys2D0; ura3D0; YIR034c::kanMX4), which has a Lys1 gene deletion and which is therefore an auxotroph for lysine, was purchased from EuroScarf. Two populations of yeast cells were grown in yeast nitrogen base liquid medium containing either 20 mg/liter normal L-lysine or 20 mg/liter L-lysine-U-13C6,15N2 (Isotec-SIGMA) for 10 generations until they reached log-phase (A600 0.7). Equal amounts of the normal and heavy SILAC-labeled yeast cells (as determined by A600 measurement) were then mixed 1:1, harvested by centrifugation for 5 min at 4,000 x g at 4 °C, washed two times with cold H2O by centrifugation, and immediately lysed for protein extraction.
Cell membranes were disrupted by boiling in a SDS solution (50 mM Tris-HCl, pH 7.5; 5% SDS; 5% glycerol; 50 mM DTT; complete protease inhibitors mixture (Roche Applied Science)). The total yeast lysate was centrifuged to remove cellular debris, the supernatant was transferred to a fresh tube, and the protein concentration in the extract was determined by Bradford assay.
1D-SDS-PAGE and In-gel Digestion of Yeast Proteins—
Yeast proteins (100 µg) were separated by one-dimensional SDS-PAGE using NuPage® Novex bis-Tris gels and NuPage® MES SDS running buffer (Invitrogen) according to the manufacturer’s instructions. The gel was stained with Coomassie Blue using the colloidal blue staining kit (Invitrogen). Protein bands were excised and digested with endoproteinase Lys-C (Wako).
Gel bands were cut into 1 mm3 cubes and washed four times with 50 mM ammonium bicarbonate, 50% ethanol. For protein reduction, gel pieces were incubated with 10 mM DTT in 50 mM ammonium bicarbonate for 1h at 56 °C. Alkylation of cysteines was performed by incubating the samples with 50 mM iodoacetamide in 50 mM ammonium bicarbonate for 1h at 25 °C in the dark. Gel pieces were washed two times with 50 mM ammonium bicarbonate, 50% acetonitrile, dehydrated with 100% ethanol, and dried in a vacuum concentrator.
The gel pieces were rehydrated with 12.5 ng/µl Lys-C in 50 mM ammonium bicarbonate and incubated for 16h at 37 °C for protein digestion. Supernatants were transferred to fresh tubes, and the remaining peptides were extracted by incubating gel pieces two times with 30% acetonitrile in 3% TFA followed by dehydration with 100% acetonitrile (MeCN). The extracts were combined and desalted using RP-C18 StageTip columns, and the eluted peptides used for mass spectrometric analysis.
NanoLC-MS/MS and Data Analysis—
All digested peptide mixtures were separated by on-line nanoLC and analyzed by electrospray tandem mass spectrometry. The experiments were performed on an Agilent 1100 nanoflow system connected to an LTQ Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). Binding and chromatographic separation of the peptides took place in a 15-cm fused silica emitter (75-µm inner diameter from Proxeon Biosystems, Odense, Denmark) in-house packed with reversed-phase ReproSil-Pur C18-AQ 3 µm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany).
The peptide mixtures were injected onto the column with a flow of 500 nl/min and subsequently gradient eluted with a flow of 250 nl/min from 5–40% MeCN in 0.5% acetic acid. Gradients were 30 min for BSA analysis and 90 min for the yeast run. The mass spectrometer was operated in the data-dependent mode to automatically switch between orbitrap-MS and orbitrap-MS/MS (MS2) acquisition. Survey full scan MS spectra (from m/z 300–1600) were acquired in the orbitrap with resolution r = 60,000 at m/z 400 (after accumulation to a target value of 1,000,000 charges in the linear ion trap). The most intense ions (up to five, depending on signal intensity) were sequentially isolated for fragmentation in the linear ion trap using collisionally induced dissociation at a target value of 100,000 charges. The resulting fragment ions were recorded in the orbitrap with resolution r = 15,000 at m/z 400.
For accurate mass measurements the lock mass option was enabled in both MS and MS/MS mode and the polydimethylcyclosiloxane (PCM) ions generated in the electrospray process from ambient air (17) (protonated (Si(CH3)2O))6; m/z = 445.120025) were used for internal recalibration in real time. For single SIM scan injections of the lock mass into the C-trap the lock mass "ion gain" was set at 10% of the target value of the full mass spectrum. The total lock mass cycle time including one orbitrap full scan and five subsequent orbitrap-MS/MS spectra was in general below 4 s. The time for the accumulation, isolation, and transfer into the C-trap of the lock mass was estimated to be a few msec at most because no time difference in cycles with and without lock mass was apparent. When calibrating in MS/MS mode the ion at m/z 429.088735 (PCM with neutral methane loss) was used instead for recalibration.
Target ions already selected for MS/MS were dynamically excluded for 30 s. General mass spectrometric conditions were: electrospray voltage, 2.4 kV; no sheath and auxiliary gas flow; ion transfer tube temperature, 125 °C; collision gas pressure, 1.3 mT; normalized collision energy, 32% for MS2. Ion selection threshold was 500 counts for MS2. An activation q = 0.25 and activation time of 30 ms was applied for MS2 acquisitions.
Peptide Identification via MASCOT Database Search—
Proteins were identified by automated database searching (Mascot Daemon, Matrix Science) against an in-house curated version of the yeast_orf (S. cerevisiae) protein sequence database or of the National Center for Bioinformatics, non-redundant database. This yeast database was complemented with frequently observed contaminants (porcine trypsin, achromobacter lyticus lysyl endopeptidase, and human keratins).
Search parameters specified an initial MS tolerance of 10 ppm and an MS/MS tolerance at 0.01 Da (minimum possible in MASCOT) and full Lys-C specificity allowing for up to 2 missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionines, N-protein acetylation, lysine-U-13C6,15N2, and N-pyroglutamate were allowed as variable modifications. Because of the high mass accuracy, the 99% significance threshold in the yeast database search was a MASCOT score of 25 even without taking into account the high fragment mass accuracy, and peptides with a greater score than this were accepted for analysis without further validation.
Averaging Precursor Masses Over the LC Profile—
To correct for peptide mass errors due to imperfect peaks caused by weak signals we have implemented a script that averages all MS full scan mass measurements of a given peptide ion over the LC peak, weighted by signal intensity. For any LTQ Orbitrap raw file containing LC-MS/MS data our script calculates all peak centroids and charge state assignments independently, making use of the lock mass to eliminate systematic error. After the peptide is recognized in adjacent survey scans, the measured peptide masses are averaged over the elution profile, weighted by its signal intensity in each scan. The program was implemented in Microsoft DotNet (C#.NET) and works with Thermo Electron Xcalibur 2.0 data files. As output, the script generates a merged MASCOT generic search-file (extension "msm") in which all peptide ion masses have been substituted with the LC-profile corrected ones. Identified peptides are listed in supplemental material 1, and peak lists for the yeast peptides displayed in the figures are given in supplemental material 3.
|
RESULTS |
|---|
|
TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSREFERENCES |
|---|
|
In the LTQ Orbitrap combination, the linear ion trap accumulates ions, isolates them, and fragments them. Although it may be possible to fragment ions in the orbitrap, it is more practical and faster to manipulate them in the linear ion trap and in the C-trap. By employing the linear ion trap as a "front end" the well known low mass cutoff for fragments applies to the LTQ Orbitrap as well. The mass range of the LTQ employed here extends to m/z 2000. Generally, fragmentation behavior in the LTQ Orbitrap will be the same as in the LTQ alone (which in turn is quite similar to three-dimensional ion traps (18, 19)) because the orbitrap solely functions as a high performance mass analyzer.
To characterize LC-MS/MS in the LTQ Orbitrap combination, we injected 50 femtomole of a tryptic digest of BSA onto a 75-µm column, performed a 30-min gradient, and measured MS and MS/MS spectra with the orbitrap as the detector. Target values were one million ions for MS ("survey") scans and 50,000 ions for MS/MS in the LTQ. Analysis of LTQ generated fragments was performed in the orbitrap. When recording the transients of the image currents, different times can be chosen, with a balance between fast acquisition cycles and high resolution. We chose "1-s survey scans," corresponding to 60,000 resolution at m/z 400 and .25-second MS/MS scans, corresponding to a resolution of 15,000. Total cycle time for one survey scan followed by MS/MS scans for up to five of the most intense ions was about 4 s on average, resulting from orbitrap transient accumulation (750 msec for MS and 190 msec each for MS/MS) and the ion fill times (Fig. 1b).
Fig. 2a shows a zoom of a typical mass spectrum. High resolving power and good signal to noise are evident. Part b of the figure contains a typical MS/MS spectrum acquired with 15,000 resolution and a target value of 50,000. The inset demonstrates that charge assignment of fragment ions is trivial given the high resolution and excellent peak shapes. Although fragmentation solely occurs in the LTQ, the user can choose to detect fragments either in the LTQ or in the orbitrap (Fig. 2c). As can be seen in the figure, the intensities of all major ions are comparable, as expected. However, the b2 ion is more abundant in the LTQ spectrum, perhaps indicating that this species fragmented further upon injection into the C-trap at the voltages chosen in our experiment. The agreement between LTQ and LTQ Orbitrap MS/MS spectra contrasts with our previous observation that on an LTQ FT certain mass ranges were better represented than others and that it was necessary to stitch three mass ranges together to obtain a representative MS/MS spectrum (20). This difference is explained by the absence of ion loss caused by time-of-flight effects over the short distance from the LTQ to the C-trap and from the C-trap to the orbitrap.
|
Finally, we observed that MS/MS spectra recorded in the orbitrap are "cleaner" (contain less noise ions) than spectra recorded in the LTQ. This is most likely caused by the high resolution of the orbitrap and its image current detection. At least 30 ions of the same species are needed for detection (14); therefore chemical background ions derived from electrospray generated solvent clusters, which are usually heterogeneous, tend not to register in the spectra. The vastly higher quality of MS/MS spectra achievable by analysis in the orbitrap will usually outweigh the longer acquisition cycle; therefore orbitrap MS/MS detection will often be preferable to LTQ MS/MS detection.
Low ppm Calibration Drift of the Orbitrap and Correction via Lock Mass—
Next we wanted to study the mass accuracy achievable on the LTQ Orbitrap. Although mass accuracy was generally excellent, we observed some drift with time, most likely caused by ambient temperature changes. It can clearly be seen in Fig. 3a that short term mass precision is much better than long term mass accuracy, which changes by a few parts per million. In mass spectrometry a lock mass is sometimes employed to compensate for such drifts and to provide an internal standard in the same spectrum containing the analyte of interest. For example, insulin was measured in the presence of a known polymer leading to a sub-ppm mass accuracy (21), and MALDI-FTICR with a dual source for analyte and internal standards resulted in similar mass accuracies (22). We used a protonated electrospray ion of polycyclodimethylsiloxane (PCM-6), which has a composition of (Si(CH3)2O))6 H+ and an exact mass of 445.120025 and is present in electrospray spectra (17). As displayed in Fig. 3b, applying a potential of a few kilovolts to an electrospray emitter generates a number of singly charged background PCM ions, which can all be used as lock masses for internal calibration during online HPLC-MS analysis. As shown in Fig. 3c, mass accuracy of the BSA peptide YLYEIAR improved from 3.7 ppm to 0.87 ppm by employing PCM as lock mass. In every mass spectrum, we utilized the lock mass information to calculate new calibration coefficients and corrected all measured m/z values essentially by applying the same ppm deviation for all masses as for the lock mass.
|
Evaluation of Lock Mass for Complex Peptide Mixtures—
Next we tested the performance of lock mass injection for the analysis of a complex peptide mixture. SILAC (23) labeled yeast was lysed, in-gel digested with Lys-C, and analyzed by LC-MS/MS on the LTQ Orbitrap. The acquisition scheme was as depicted in Fig. 1b, using lock mass injection for all spectra. After identification of yeast peptides by database searching, mass differences between calculated and measured peptide masses were binned in 0.5-ppm windows. Fig. 4a shows that the distribution of mass errors is roughly Gaussian with almost all errors within plus or minus two ppm. To investigate the cause of the few outliers at 3–5-ppm mass deviation, we plotted the mass deviation as a function of peptide intensity. Fig. 4b suggests that the outliers are mainly caused by low signal. Makarov et al. (14) have reported previously that signal to noise is a determinant of achieved mass accuracy in orbitrap mass spectrometry, which is also in agreement with previous observations on other instruments (24).
|
Therefore we implemented a script to follow precursors over the LC peaks, which averages the mass measurements weighted by signal intensity. Fig. 5 shows the results of these calculations. As expected, mass accuracy was further improved and was generally within a 1 ppm absolute deviation from the calculated values. Fig. 5b demonstrates that all peptides after the LC-profile correction have a maximal mass error of less than 2 ppm (absolute average mass accuracy of 0.48 ppm and a standard deviation of 0.38 ppm).
|
|
Dependence of achieved mass accuracy on signal or signal to noise is not unique to the LTQ Orbitrap. Unfortunately, in current proteomic practice, a "least common denominator" strategy is pursued when searching databases. The precursor and fragment mass tolerances are set to encompass even the worst mass measurements. After systematic mass errors have been removed by iterative recalibration (31) or by lock mass as shown here, a better strategy would be to specify very tight mass tolerances for well defined peaks and larger mass tolerances for weak peaks. Such a strategy could be implemented in a second step, after all peptides have been searched with a larger mass tolerance. In this second step peptides with good signal but large deviation from the calculated masses would be removed from consideration, or at least obtain a reduced score. Ultimately, each precursor mass measurement should be accompanied by its own standard deviation. Statistical evaluation tools such as PeptideProphet (32) can already accommodate such additional evidence in a quantitative way.
The lock mass employed here, PCM, is ubiquitously present in laboratory air. However, other background ions could be used as well, and it is also possible to use more than one lock mass in the same spectrum.
Lock mass injection made use of the intermediate storage compartment provided by the C-trap. Although we have used the C-trap for storing a constant number of lock mass ions, it clearly can be used for many other interesting purposes as well. For example, the C-trap could be filled rapidly with several narrow mass ranges of interest or several MS/MS fragment ions generated from different precursor ions before high resolution analysis of the accumulated ions in the orbitrap.
In comparison with the LTQ-FTICR, the LTQ Orbitrap is capable of similar mass accuracy, as shown here. However, here we achieve the high mass accuracy with the help of a lock mass whereas in the LTQ-FTICR we used "SIM scans," the isolation and injection of a narrow mass range into the ICR cell (3). An advantage of the lock mass strategy is that no extra time needs to be spent on SIM scans. Moreover, when the LTQ-FTICR is operated without SIM scans and with a high ion load at the limit of its space charge capacity to maximize dynamic range, then mass accuracy is about an order of magnitude worse than what we demonstrate here for the LTQ Orbitrap.
What is the utility of the robust, high mass accuracy demonstrated here? Zubarev et al. (33) have pointed out that a mass accuracy of 1 ppm could constrain peptide candidates to just a few sequences and thereby greatly facilitate peptide identification. Smith and co-workers added a retention time criterion and suggested that such "accurate mass and time tags" could be sufficient for peptide identification (34). This concept has been difficult to evaluate because mass accuracies actually achieved so far seem to have been relatively modest (see for example, Ref. 35). Even with the accuracy reported here, we do not believe that the mass alone is sufficient to identify peptides in typical proteomic experiments given the complexities introduced by incomplete cleavage and peptide modifications. However, for specialized situations, such as re-analysis of subproteomes, the number of candidate peptide sequences will be very small, and even low accuracy tandem mass spectra can then unequivocally decide if the candidate peptide is indeed present. Likewise, this mass accuracy will be extremely helpful in the elucidation of post-translational modifications, which presents a special problem because of the "combinatorial explosion" caused when considering many different types of modifications (36).
In conclusion, we have shown that a new compact hybrid mass spectrometer, the LTQ Orbitrap, is capable of very high mass accuracy using a lock mass strategy. High mass accuracy is routinely achievable in both the MS and MS/MS mode, and alone or in combination with MS3 strategies (37) should help to virtually eliminate the problem of false positive peptide identification in proteomics and to identify post-translational modifications much more easily than currently possible.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Published, MCP Papers in Press, October 24, 2005, DOI 10.1074/mcp.T500030-MCP200
1 The abbreviations used are: SIM, selected ion monitoring; MS/MS, tandem MS; LTQ, Thermo Electron linear quadrupole ion trap; RF, radio frequency; SILAC, stable isotope labeling by amino acids in cell culture; PCM, polycyclodimethylsiloxane. 
* Work at the Center for Experimental BioInformatics is supported by a generous grant by the Danish National Research Foundation. Work on this project at the Max-Planck-Institute of Biochemistry and Thermo Electron was supported by "Interaction Proteome," a 6th Framework grant by the European Union research directorate. 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.
** To whom correspondence should be addressed: Dept. of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, D-82512 Martinsried, Germany. E-mail: mmann{at}biochem.mpg.de
|
REFERENCES |
|---|
|
TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Trinkle-Mulcahy, S. Boulon, Y. W. Lam, R. Urcia, F.-M. Boisvert, F. Vandermoere, N. A. Morrice, S. Swift, U. Rothbauer, H. Leonhardt, et al. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes J. Cell Biol., October 20, 2008; 183(2): 223 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Majeran, B. Zybailov, A. J. Ytterberg, J. Dunsmore, Q. Sun, and K. J. van Wijk Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells Mol. Cell. Proteomics, September 1, 2008; 7(9): 1609 - 1638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bantscheff, M. Boesche, D. Eberhard, T. Matthieson, G. Sweetman, and B. Kuster Robust and Sensitive iTRAQ Quantification on an LTQ Orbitrap Mass Spectrometer Mol. Cell. Proteomics, September 1, 2008; 7(9): 1702 - 1713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Mertins, H. C. Eberl, J. Renkawitz, J. V. Olsen, M. L. Tremblay, M. Mann, A. Ullrich, and H. Daub Investigation of Protein-tyrosine Phosphatase 1B Function by Quantitative Proteomics Mol. Cell. Proteomics, September 1, 2008; 7(9): 1763 - 1777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. F. Waanders, R. Almeida, S. Prosser, J. Cox, D. Eikel, M. H. Allen, G. A. Schultz, and M. Mann A Novel Chromatographic Method Allows On-line Reanalysis of the Proteome Mol. Cell. Proteomics, August 1, 2008; 7(8): 1452 - 1459. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Shin, H.-J. Jung, S.-W. Hyung, H. Kim, D. Lee, C. Lee, M.-H. Yu, and S.-W. Lee Postexperiment Monoisotopic Mass Filtering and Refinement (PE-MMR) of Tandem Mass Spectrometric Data Increases Accuracy of Peptide Identification in LC/MS/MS Mol. Cell. Proteomics, June 1, 2008; 7(6): 1124 - 1134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Maitra, C.-F. Chou, C. A. Luber, K.-Y. Lee, M. Mann, and C.-Y. Chen The AU-rich element mRNA decay-promoting activity of BRF1 is regulated by mitogen-activated protein kinase-activated protein kinase 2 RNA, May 1, 2008; 14(5): 950 - 959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Graumann, N. C. Hubner, J. B. Kim, K. Ko, M. Moser, C. Kumar, J. Cox, H. Scholer, and M. Mann Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Proteome Quantitation of Mouse Embryonic Stem Cells to a Depth of 5,111 Proteins Mol. Cell. Proteomics, April 1, 2008; 7(4): 672 - 683. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Karunakaran, J. Rey-Ladino, N. Stoynov, K. Berg, C. Shen, X. Jiang, B. R. Gabel, H. Yu, L. J. Foster, and R. C. Brunham Immunoproteomic Discovery of Novel T Cell Antigens from the Obligate Intracellular Pathogen Chlamydia J. Immunol., February 15, 2008; 180(4): 2459 - 2465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Macek, F. Gnad, B. Soufi, C. Kumar, J. V. Olsen, I. Mijakovic, and M. Mann Phosphoproteome Analysis of E. coli Reveals Evolutionary Conservation of Bacterial Ser/Thr/Tyr Phosphorylation Mol. Cell. Proteomics, February 1, 2008; 7(2): 299 - 307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Matic, M. van Hagen, J. Schimmel, B. Macek, S. C. Ogg, M. H. Tatham, R. T. Hay, A. I. Lamond, M. Mann, and A. C. O. Vertegaal In Vivo Identification of Human Small Ubiquitin-like Modifier Polymerization Sites by High Accuracy Mass Spectrometry and an in Vitro to in Vivo Strategy Mol. Cell. Proteomics, January 1, 2008; 7(1): 132 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
