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, 48 (3), 311-9

Comprehensive Proteomic Analysis of Schizosaccharomyces Pombe by Two-Dimensional HPLC-tandem Mass Spectrometry

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Comprehensive Proteomic Analysis of Schizosaccharomyces Pombe by Two-Dimensional HPLC-tandem Mass Spectrometry

Laurence M Brill et al. Methods.

Abstract

We describe a detailed and widely applicable method for comprehensive proteomic profiling of the fission yeast Schizosaccharomyces pombe by 2-dimensional high performance liquid chromatography-electrospray ionization-tandem mass spectrometry that demonstrates high sensitivity and robust operation. Steps ranging from the preparation of total proteins, digestion of proteins to peptides, and separation of peptides by two-dimensional (1. strong cation exchange and 2. reversed-phase) high performance liquid chromatography followed by tandem mass spectrometry and data processing have been optimized for our instrumentation platform. Using this technology, we identify ca. 3400 proteins per sample and have identified an estimated 4600 proteins in vegetative cells (equal to ca. 90% of the predicted S. pombe proteome) at a false discovery rate of 0.02. Considering the fact that approximately 500 genes are strongly induced during sexual differentiation, and sexual differentiation was not included in our experiments, the proteomic profiling technique affords what should be virtually complete coverage of the vegetative S. pombe proteome. In addition, these methods are widely applicable, having been used for proteomic profiling of several other organisms.

Figures

Figure 1
Figure 1
Instrumentation platform used in the proteomic analyses. (A) Sample injection, from the syringe mounted on the autosampler, into the sample injection port (red arrow). Samples for SCX and RP separations are introduced into the HPLC at this point. (B) Offline collection of SCX fractions using the fraction collection tool (blue and tan), which is magnetically suspended from the autosampler arm (black cylinder). The SCX eluate drips into open vials (red arrow). (C) Syringe drawing up a portion of a SCX fraction (black arrow) for subsequent re-injection into the sample injection port (shown in panel A), for RP HPLC-MS/MS analysis. Scale bars represent 2.5 cm (A–C). (D) Composite instrumentation platform, including the UV detector (UV); solvent bottle holder on top of the module controlling gas flow and choice of 40:40:20 isoproponal:acetonitrile:water (40:40:20 wash solvent) or HPLC solvents (solv.); HPLC pumps/mixers/splitter for solvents A/B and solvents C/D (pumps A/B use a splitter but C/D do not) and 2 valves (HPLC) with the necessary plumbing (PEEK and PEEKsil tubing), SCX and immobilized metal affinity chromatography (IMAC) columns attached (the IMAC application for phosphopeptide enrichment will be described elsewhere); the sample loop mounted on HPLC valve 1 (loop); the sample injection port (port); polymeric trap for peptide capture and desalting (RP-pep. capture) on the valve that is mounted on the mass spectrometer; the RP analytical column (RP column) mounted in the ESI source (ESI); the autosampler/fraction collector containing refrigerated drawers for reagent, sample and SCX fraction storage; and the hybrid linear ion trap- (ion trap) OrbitrapXL (Orbitrap) mass spectrometer; scale bar represents 10 cm.
Figure 2
Figure 2
HPLC gradient profiles for the SCX separation (A) and RP separation (B) of peptides are shown. For the SCX separation, solvents C and D are used, and for the RP separation, solvents A and B are used. Solvent composition is described in the text.
Figure 3
Figure 3
Simplified schematic diagram of the plumbing for liquid flow through the HPLC, autosampler, UV detector flow cell, SCX and RP columns. The flow paths that are used for the separations described in this article are all shown. All tubing is 1/16” outer diameter (OD) PEEK, with varied inner diameters, with the exception that PEEKsil tubing (also 1/16” OD) is used for all sample flow paths. Tubing was obtained primarily from Michrom and also from Upchurch Scientific (Seattle, WA). (A) Flow paths for sample introduction and SCX column stabilization, immediately prior to SCX separation are shown. Abbreviations: ASV, autosampler valve; HPLC V1, HPLC valve 1; mix 2, mixer 2 (for solvents C and D); HPLC V2, HPLC valve 2; UV, UV detector flow cell. Two flow paths, marked by arrows, are active at this stage: 1) Flow of the sample, from the syringe, for sample introduction into the (100 μl) sample loop, and 2) flow from pumps C and D (not shown for clarity), through mixer 2, for stabilization of solvent flow and pressure through the SCX column prior to the SCX separation. (B) Flow path during the SCX separation, which no longer involves sample introduction. To enter this stage following that shown in panel A, HPLC valve 1 switches, resulting in reversal of the sample flow back out of the sample loop and onto the SCX column. Concurrent to the switching of valve 1, the autosampler automatically suspends the fraction collection tool above the vials (Fig. 1B), in order to collect the eluate from the SCX column. The fraction collection tool is automatically moved to a fresh vial every 2.0 min, resulting in collection of 24 × 400 μl fractions. The SCX gradient, consisting of solvents C and D (see the text and Fig. 2A), is applied, resulting in peptide separation throughout the gradient. (C) Flow paths for sample introduction immediately prior to reversed phase (RP) separation; abbreviations: MSV, valve mounted on the mass spectrometer; mix 1, mixer 1 (for solvents A and B); cp tp, capillary peptide trap (polymeric); and AC, C18 reversed-phase, capillary-scale (1/16″ OD) analytical column. Two flow paths, marked by arrows, are active at this stage: 1) Flow of the sample, from the syringe, for sample introduction onto the peptide trap (cp tp) followed by desalting of the captured peptides, and 2) flow from pumps A and B (not shown for clarity), through mixer 1, for stabilization of solvent flow and pressure through the C18 analytical column (AC). The high voltage (1.8 kV) is manually activated at the start of this stage using the tune page, which is part of the control software for the mass spectrometer. (D) Flow path during the reversed-phase separation, which no longer involves sample introduction. To enter this stage following that shown in panel C, the valve mounted on the mass spectrometer switches, resulting in reversal of the solvent flow back through the peptide trap, gradient elution of the peptides off of the peptide trap, their separation in the analytical column, elution, and ionization in the ESI source, just prior to introduction into the mass spectrometer.
Figure 4
Figure 4
Representative results of a 2D HPLC-MS/MS analysis of the S. pombe proteome. (A) Typical trace of a SCX separation of tryptic peptides; A214 = absorbance at 214 nm. (B) Total ion chromatogram (TIC) resulting from re-injection of 10% of SCX fraction 2 and (C) SCX fraction 7 for RP HPLC-MS/MS analysis. The number of counts recorded by the mass spectrometer, in arbitrary units (Thermo Fisher Scientific), is shown. (D) The entire m/z range (the instrument is programmed to scan from m/z 300 to 2,000, but most scans contain a portion of this range) of an ESI mass spectrum resulting from re-injection of SCX fraction 7, at an RP retention time of 110.40 min. (E) The m/z range from 918 to 936 of the same ESI mass spectrum as in (D). Note the doubly charged ion with a measured monoisotopic m/z of 925.8990 and a theoretical monoisotopic m/z of 925.9007, which demonstrated 1.8 ppm mass accuracy in this scan for this ion. The isotopes of this same ion, with an increased mass due to the natural incorporation of 1, 2, 3 or 4 heavy atoms are also labeled. (F) MS/MS spectrum resulting from collision-induced dissociation (CID) of the precursor ion with an m/z of 925.8990 (shown in panel E). This MS/MS spectrum, when searched against the S. pombe proteome, demonstrated that the precursor ion (measured m/z of 925.8990) was derived from the pub1 ubiquitin protein ligase E3 (accession number SPAC11G7.02). b- and y product ions obtained upon CID are labeled, as are sites in the peptide sequence that were detectably fragmented via CID.
Figure 5
Figure 5
Error, sensitivity and number of proteins are shown for protein identification vs. minimum protein probability, as predicted by ProteinProphet®, for the MS/MS data resulting from analysis of a typical sample of the total proteome of S. pombe. (A) Error (e) and sensitivity (s) are shown for injection of 10% of SCX fraction 2 onto RP media, separation using the RP gradient, coupled to ESI-MS/MS (SCX f2->RP HPLC-MS/MS); (B) the same analysis using SCX fraction 7 (SCX f7->RP HPLC-MS/MS) or the same analysis using the data from SCX fractions 1–24 (all fractions) searched together in a composite search of all the data from the sample (C; SCX f1-SCX f24->RP HPLC-MS/MS). (D) The ProteinProphet® Sensitivity/Error Info shows the protein identification statistics from a database search of the composite dataset (re-injection of SCX fractions 1–24) for the sample as a function of minimum protein probability (Min. prob.). The sensitivity of protein identification (Sens.) is the number of proteins identified divided by the total predicted number of correct proteins (3435 for this data set); error (Err.) is the estimated protein FDR at the given minimum protein probabilities; number correct (Num. corr.) = estimated number of correct protein identifications at the given minimum protein probabilities and number incorrect (Num. incorr.) = estimated number of incorrect protein identifications at the given minimum protein probabilities. At a minimum protein probability of 0.50 (0.50*), the FDR is estimated at 0.020 for this data set.
Figure 6
Figure 6
Representative view of QTools output showing spectral counts of S. pombe proteins from 4 different samples in an experiment, each of which was subjected to 2D HPLC-MS/MS analyses. The protein identifier and description were derived from the S. pombe protein database (described in the text), and the numbers in the sample (Sam.) columns are the number of spectral counts from all of the peptides belonging to the identified proteins. The biological processes, cellular components and molecular functions, when known, are also shown.

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