Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 28

6. Developing solution: Dissolve 30 g of K 2 CO 3 and4mgofNa 2 S 2 O 3 ·5H 2 Oin1L
deionized water. Prior to use, add 500 μL 37% formaldehyde to 1 L developing
solution.
7. Stop solution: 1% (w/v) glycine dissolved in deionized water.
8. Storage Phosphor Screens 20 cm × 25 cm (Molecular Dynamics, Krefeld,
Germany).
9. Storm 840 Phosphor Imager (Molecular Dynamics).
10. Typhoon laser scanner (9200, 9210, 9400, or 9410).
11. Scanner X finity ultra (Quato Graphic, Braunschweig, Germany).
2.5. In-Gel Digestion
1. Spot cutter (Proteome Work™) with a picker head of 2 mm (GE-Healthcare).
2. Ettan Spot Handling Workstation (GE-Healthcare).
3. 50 m M ammonium bicarbonate/50% (v/v) methanol.
4. 75% (v/v) acetonitrile.
5. Trypsin solution: 1 mg/mL mg trypsin dissolved (Promega, Madison, WI, USA)
in 20 m M ammonium carbonate (freshly prepared).
6. 50% (v/v) acetonitrile/0.1% (w/v) trifluoroacetic acid (TFA).
7. Matrix solution 50% (v/v) acetonitrile/0.5% TFA saturated with -cyano-4-
hydroxycinnamic acid (CHCA).
8. 0.5% (w/v) TFA/50% (v/v) acetonitrile.
2.6. Protein Identification by Mass Spectrometry
1. Proteome-Analyzer 4700 (Applied Biosystems, Foster City, CA, USA).
2. 4700 Explorer™ software.
3. Genome sequences of S. aureus in FASTA format (www.ncbi.nlm.nih.gov).
3. Methods
In order to investigate the physiology and virulence of S. aureus , we sought
to analyze the cytoplasmic and extracellular proteome of S. aureus by using 2D
gel analysis combined with MALDI-TOF MS/MS ( see Fig. 1 and Note 2 ). For
low-complexity organisms such as bacteria, the majority of cellular and extra-
cellular proteins can be visualized within the main analytical window of pHs
ranging from 4-7 (cytoplasmic proteins) or 3-10 (extracellular proteins); thus,
gel-based proteomics will remain an extremely valuable tool, because it econom-
ically permits the comparative and quantitative protein profiling in multi-sample
comparisons. Protein master gels showing most of the proteins predicted for the
respective cell compartment (e.g., cytosol, supernatant) ( see Fig. 1 ) offer the
chance to study the regulation of the synthesis and/or the amount of all these
proteins under various conditions and in different mutant strains. In this way,
the 2D protein pattern of cells grown under various growth restricting conditions