Ole Vorm

System-wide Perturbation Analysis with Nearly Complete Coverage of the Yeast Proteome by Single-shot Ultra HPLC Runs on a Bench Top Orbitrap

Yeast remains an important model for systems biology and for evaluating proteomics strategies. In-depth shotgun proteomics studies have reached nearly comprehensive coverage, and rapid, targeted approaches have been developed for this organism. Recently, we demonstrated that single LC-MS/MS analysis using long columns and gradients coupled to a linear ion trap Orbitrap instrument had an unexpectedly large dynamic range of protein identification (Thakur, S. S., Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M. (2011) Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here we couple an ultra high pressure liquid chromatography system to a novel bench top Orbitrap mass spectrometer (Q Exactive) with the goal of nearly complete, rapid, and robust analysis of the yeast proteome. Single runs of filter-aided sample preparation (FASP)-prepared and LysC-digested yeast cell lysates identified an average of 3923 proteins. Combined analysis of six single runs improved these values to more than 4000 identified proteins/run, close to the total number of proteins expressed under standard conditions, with median sequence coverage of 23%. Because of the absence of fractionation steps, only minuscule amounts of sample are required. Thus the yeast model proteome can now largely be covered within a few hours of measurement time and at high sensitivity. Median coverage of proteins in Kyoto Encyclopedia of Genes and Genomes pathways with at least 10 members was 88%, and pathways not covered were not expected to be active under the conditions used. To study perturbations of the yeast proteome, we developed an external, heavy lysine-labeled SILAC yeast standard representing different proteome states. This spike-in standard was employed to measure the heat shock response of the yeast proteome. Bioinformatic analysis of the heat shock response revealed that translation-related functions were down-regulated prominently, including nucleolar processes. Conversely, stress-related pathways were up-regulated. The proteomic technology described here is straightforward, rapid, and robust, potentially enabling widespread use in the yeast and other biological research communities.

Improved mass accuracy in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides

One problem of matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry is the moderate mass accuracy that typically can be obtained in routine applications, Here we report improved mass accuracy for peptides, even when low amounts and complex peptide mixtures are used. A new procedure for preparing matrix surfaces is used, and there is no need to mix the matrix with the sample or to add internal standards. Examples are shown with a mass accuracy better than 50 ppm in a peptide mixture. Peptide mapping as well as sequencing by creating “ragged ends” or “ladder sequencing” should benefit especially from the improved mass accuracy.

Detector Bias Gating for Improved Detector Response and Calibration in Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Simple circuitry was developed for detector bias grating as an effective and convenient scheme for conserving the charge in detectors used in mass-analysed laser desorption/ionization time-of-flight mass spectrometry. An enhanced detector response by a factor of 3-10 was observed by detector gating compared with the commonly used low-mass deflection. Further, detector gating allowed the acquisition of the low-mass region of the time-of-flight spectrum at reduced sensitivity. Thus the analyte signal and matrix signals could be obtained in the same spectrum with an excellent signal-to-noise ratio and without exceeding the dynamic range of the digitizer. This feature, along with the recently described sample preparation procedure by fast evaporation, yields matrix ion signals that are very well defined and can be used for highly accurate spectrum calibration. Together, these developments open the way to routine mass determinations with errors in the sub-50 ppm range without adding internal standards.

Synthesis and characterization of a 25-residue rubredoxin(II)-like metalloprotein and its valine-leucine mutant

An iron‐sulfur metalloprotein containing the 5–12 and 35–50 residues of Desulfovlbrio gigas rubredoxin has been synthesized by Fmoc solid phase peptide synthesis and subsequent peptide folding. A Gly links the two residue chains between Val‐5 and Glu‐50. Sybyl Tripos structure optimization indicates only minor structural changes of the folded synthetic protein compared to the similar residue positions in the native protein. The UV‐VIS spectrum of the reduced synthetic protein is very similar to that or native D. gigas rubredoxin and the molecular mass determined by laser mass spectrometry has the expected value (± 2D). No metal is transferred to the gas phase by the laser beam merely by mixing the peptide and iron(II), substantiating that the folding procedure is a necessary pre‐requisite for protein formation. The Val → Leu41 chemical mutant has also been synthesized and behaves in a closely similar fashion.