The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for its application in microbial physiology. DNA arrays were used to calculate the number of genes transcribed in growing cells. From the 4100 B. subtilis genes, 2515 were actively transcribed in cells grown under standard conditions. From these genes 1544 proteins should be covered by our standard gel system pI 4-7. Using this standard gel system and supplementary zoom gels (pI 5.5-6.7, 5-6, 4.5-5.5, and 4-5) 693 proteins which are expressed in growing cells were detected that cover more than 40% of the vegetative proteome predicted for this region. Particularly broad coverage and thus comprehensive monitoring will be possible for central carbohydrate metabolism (glycolysis, pentose phosphate shunt, and citric acid cycle), amino acid synthesis pathways, purine and pyrimidine metabolism, fatty acid metabolism, and main cellular functions like replication, transcription, translation, and cell wall synthesis. Comparing the theoretical pI and Mr values with those experimentally determined a reasonable correlation was found for the majority of protein spots. By a color code outliers with dramatic deviations in charge or mass were visualized that may indicate post-translational modifications. In addition to the cytosolic neutral and alkaline proteins, 130 membrane proteins were found relying on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) techniques. The vegetative proteome containing 876 proteins in total is now ready for physiological applications. Two main proteome fractions (pI 4-7 and zoom gel pI 4.5-5.5) should be sufficient for such high-throughput physiological proteomics.

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is an excellent experimental tool for physiological studies opening a new perspective for understanding overall cell physiology. This is the intriguing outcome of a method introduced by Klose and O'Farrell independently 25 years ago. Physiological proteomics requires a 2-D reference map on which most of the main proteins were identified. In this paper, we present such a reference map with more than 300 entries for Bacillus subtilis proteins with an isoelectric point (pI) between 4 and 7. The most abundant proteins of exponentially growing cells were compiled and shown to perform mainly housekeeping functions in glycolysis, tricarboxylic acid cycle (TCC), amino acid biosynthesis and translation as well as protein quality control. Furthermore, putative post-translational modifications were shown at a large scale, with 47 proteins in total forming more than one spot. In a few selected cases evidence for phosphorylation of these proteins is presented. The proteome analysis in the standard pI range was complemented by either stretching the most crowded regions in a narrow pH gradient 4.5-5.5, or by adding other fractions of the total B. subtilis proteome such as alkaline proteins as well as extracellular proteins. A big challenge for future studies is to provide an experimental protocol covering the fraction of intrinsic membrane proteins that almost totally escaped detection by the experimental procedure used in this study.

(2) is undoubtedly still the gold standard to separate complex protein mixtures. This technique allows the mass spectrometric identification of hundreds of proteins after their separation on a two-dimensional (2D) 1 gel, thereby covering an essential portion of "low complexity" proteomes such as those of bacteria. However, there are limitations to 2D PAGE that make certain classes of proteins inaccessible. Gel-critical properties include extremes in pI and molecular mass, but the most significant shortcoming is certainly the poor separation of proteins showing a pronounced hydrophobicity. The dynamic range in protein concentration that can be covered by 2D PAGE regarding non-radioactive staining methods spans 3-4 orders of magnitude, whereas the protein concentration in human blood serum extends at least 9 orders (3). For this reason a simple 2D gel approach is insufficient to analyze entire proteomes, including very low abundance proteins. The challenges inherent to a gel-based approach point to a demand for alternative techniques.Although the characterization and quantitation of stained protein spots on 2D gels was introduced decades ago and further developed to date (4 -6), attempts of protein identification and concurrent quantitation exclusively based on mass spectrometry have emerged over the last years (7). Initially the combination of multidimensional chromatography and tandem mass spectrometry that became known as shotgun proteomics was used to identify hundreds of proteins out of highly complex peptide mixtures (8 -12). Very soon the first gel-free methods arose allowing a relative quantitation of proteins from different samples. A common technique is the use of stable isotope labeling of proteins or peptides, mostly realized by chemical linking of tag and biomolecule. Here one of the sample sets is provided with a "light" tag, whereas the others are linked to heavy isotope-enriched variants of the tag. Although almost all of the previously established methods (13-15) make use of a quantitation procedure based on ion signal intensity observed at the MS level, the recently introduced isobaric tagging for relative and absolute quantitation (iTRAQ TM ) supports a quantitation based on reporter ion signals observed at the MS/MS level that is linked with several advantages (16). First of all, the differential labeling of peptides does not challenge scan rates of mass spectrometers because the complexity at the MS level is not increased. Second, the detection of peptides originating from low abundance proteins is facilitated by the addition of ion currents of equal but differentially labeled peptides in MS spectra. On the one hand the requirement of MS/MS experiments only allows a quantitation of peptide signals exceeding a given threshold, but on the other hand the unambiguous identification of a peptide becomes more likely because it is not only based on 1 The abbreviations used are: 2D, two-dimensional; 1D, one-dimensional; C.I., confidence interval; SCX, strong cation exchange; iTRAQ, isobaric tagging f...

In the growing field of systems biology, the knowledge of protein concentrations is highly required to truly understand metabolic and adaptational networks within the cells. Therefore we established a workflow relying on long chromatographic separation and mass spectrometric analysis by data independent, parallel fragmentation of all precursor ions at the same time (LC/MS E ). By prevention of discrimination of co-eluting low and high abundant peptides a high average sequence coverage of 40% could be achieved, resulting in identification of almost half of the predicted cytosolic proteome of the Gram-positive model organism Bacillus subtilis (>1,050 proteins). Absolute quantification was achieved by correlation of average MS signal intensities of the three most intense peptides of a protein to the signal intensity of a spiked standard protein digest. Comparative analysis with heavily labeled peptides (AQUA approach) showed the use of only one standard digest is sufficient for global quantification.The quantification results covered almost four orders of magnitude, ranging roughly from 10 to 150,000 copies per cell. To prove this method for its biological relevance selected physiological aspects of B. subtilis cells grown under conditions requiring either amino acid synthesis or alternatively amino acid degradation were analyzed. This allowed both in particular the validation of the adjustment of protein levels by known regulatory events and in general a perspective of new insights into bacterial physiology. Within new findings the analysis of "protein costs" of cellular processes is extremely important. Such a comprehensive and detailed characterization of cellular protein concentrations based on data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MS E ) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities. Molecular & Cellular

Successful proteome analyses of highly dilute samples are strongly dependent on optimized workflows considering especially sample preparation prior to highly sensitive mass spectrometric analysis. Various methods are available for enrichment of proteome samples, each characterized by specific advantages and disadvantages limiting their general application as a method of choice. Here we suggest an optimized universal protocol ensuring reproducibility and effective enrichment of dilute samples by commercial affinity beads. By comparably assessing the performance of the new protocol with selected standard enrichment techniques, we show the seamless application of the enrichment in common mass spectrometry based proteomic workflows. Further, novel applications are suggested including a facile storage and shipping of desiccated, trapped proteome samples at ambient temperatures and usage of the affinity beads for gel-free proteomic approaches.