Table II. Yeast organellar proteomics studies.
A summary of the 18 experimental proteomes discussed in this review is given. 2-DE, two-dimensional gel electrophoresis; BAC, N,N′-bisacrylylcystamine; CTAB, cetyltrimethylammonium bromide; GC, gas chromatography; HA, hemagglutinin; nLC, nanoflow LC; LDS, lithium dodecyl sulfate; MudPIT, multidimensional protein identification technology; PFP, peptide fingerprint; RP, reverse-phase; SAX, strong anion exchanger; SCX, strong cation exchanger; WB, Western blot; FFE, free flow electrophoresis; 1D, one-dimensional; 2D, two-dimensional; μLC, microcapillary LC; PMF, peptide mass fingerprint; EC, exchange chromatography; TMDs, transmembrane domains.
| Organelle | Straina and growth conditionsb | Spheroplasting and lysis | Purification separation enrichment | Stripping | Purity assessment | Protein and/or peptide separation | MS analysis | No. of identified proteins (%TMDs) | Detected abundance range | Validation | Ref. |
|---|---|---|---|---|---|---|---|---|---|---|---|
| copies/cell | |||||||||||
| Mitochondria | CW04 (n), YPLc | Zymolyase; osmotic lysis | Differential centrifugation | No | EM | SDS-PAGE | nLC-ESI-MS/MS | 179 (12%) | 149–882,841 | GFP or HA tag, computation | 27 |
| Mitochondria | YPH499 (n), YPGd | Zymolyase; mechanical disruption | 1) Differential centrifugation, 2) sucrose density gradient (32/60% interface) | Salt, shaving with trypsin | WB | 1) 2-DE, 2) SCX-RP-nLC,e 3) SDS-PAGEe | 1) MALDI-MS/MS or nLC-ESI, 2) ESI-MS, 3) nLC-ESI-MS/MS | 750 (18%) | 99–1,255,722 | Protein import assay, presequence | 28 |
| Mitochondria | BJ1991 (n), YPD versus YPG | Zymolyase; mechanical disruption | Sucrose density gradient | EDTA | 2-DE | PFP | 251 (6%) | 149–1,018,216 | Transcriptome | 34 | |
| Mitochondria | W303 (n), S1001, YPL, YPD, SCL, and SCDf | 1) Nycodenz density gradient, 2) FFE, 3) differential centrifugation | Denaturation in 7 m urea, 2 m thiourea, 1% CHAPS | EM | RP-LC | ESI-MS/MS, LC-FTICR | 546 (17%) | 57–1,255,722 | Co-enrichment with known marker using WB | 26 | |
| Mitochondria | YPH499 (n), YPG | Zymolyase; mechanical disruption | 1) Differential centrifugation, 2) sucrose density gradient (32/60% interface)g | EDTA | WB | 1) 2-DE, 2) SAX- or SCX-RP-LC,e 3) SDS-PAGE | 1) MALDI-MS/MS or nLC-ESI, 2) ESI-MS, 3) nLC-ESI-MS/MS | 851 (18%) | 99–1,255,722 | 30 | |
| Mitochondria | YPH499 (n), YPG | Zymolyase; mechanical disruption | Intact mitochondria as in Ref. 28, Outer membranes: sucrose density gradient (interface 15/32%) | EDTA, trypsin | WB | 1) 2D BAC/SDS, 2) SCX-RP-LC,e 3) SDS-PAGE | 1) MALDI-MS/MS, 2) ESI-MS/MS, 3) nLC-ESI-MS/MS | 117 (20%) | 147–882,841 | Computational and biochemical analysis | 29 |
| Peroxisomes | BJ1991 (n), BJ1991Δpox1, YNOh | Lyticase | Sucrose density gradient (47–68%) | Salt, pH 11.3 | 1D SDS-PAGE | μLC- and nLC-ESI-MS/MS | 45 (18%) | 358–1,255,722 | No | 22 | |
| Peroxisomes | BY4743 (2n), SCIMi | Zymolyase; mechanical disruption | 1) Differential centrifugation, 2) Nycodenz density gradient (35–50% interface) | EDTA | μLC | GC-ESI-MS/MS | 44 (11%) | 238–111,935 | 23 | ||
| Peroxisomes | BY4743 (2n), SCIMi | Zymolyase; mechanical disruption | 1) Differential centrifugation, 2) Nycodenz density gradient (35–50% interface) | pH 11.3 | WB | ICAT-μLC | ESI-MS/MS | 72 (21%) | 238- 268,717 | GFP tagging, mutant phenotype | 21 |
| Lipid particles | X2180-1A (n), FY1679 (n, 2n), YPD | Zymolyase; osmotic lysis | Ficoll density gradient | Delipidation | Enzymatic assays, EM | 1D SDS-PAGE | nLC-ESI-MS/MS | 18 (28%) | 1,212–168,876 | Lipid and sterol composition in deletion mutants | 31 |
| Nucleus | BY 4741 (n), YPD | Zymolyase; mechanical disruption | DNase I, heparin, sucrose density gradient | SCX-RP-nLC | ESI-MS/MS | 2698 (11%) | 49–1,255,722 | 9 | |||
| Nucleus | YPD (Wickerham), BY 4743 (2n) | Zymolyase, lyticase, Glucanex; mechanical disruption | DNase I, heparin, sucrose density gradient | 1) Peptide IEF, 2) SCX-RP-nLC, 3) 1D SDS-PAGE, 4) phosphocellulose chromatography + SDS-PAGE | ESI-MS/MS | 1889 (5%) | 41–1,255,722 | 10 | |||
| Golgi | CJY119 (n), HIY1 (n), YPD | Lyticase; osmotic shock | 1) Differential centrifugation, 2) immunoprecipitation | Triton X-114 phase separation | WB | 1D SDS-PAGE | MALDI-MS/MS | 52 (48%) | 238–82,660 | Knock-out mutant analysis; HA tagging | 6 |
| Vacuolar lumen | SEY6210 (n), YPD | Zymolyase; mechanical disruption | Sucrose/sorbitol/Ficoll density gradient | Proteinase K | WB, marker enzymes assays, microscopy, flow cytometry | 2-DE | MALDI-MS/MS, nLC-IT-MS/MS, ESI-MS/MS | 118 (5%) | 172–1,018,216 | Internal | 8 |
| Vacuolar membrane | W303 (n), YPD | Zymolyase, lyticase, Glucanex; osmotic lysis | Ficoll density gradient | EDTA, pH | WB | SCX-RP-nLC | MALDI-MS/MS | 148 (57%) | 57–314,034 | LOPIT | 7 |
| Cell wall | FY833 (n), YPD | Direct mechanical disruption | Differential centrifugation, HF-pyridine extraction | Salt, EDTA, SDS, NaOH | No information | Anion-exchange chromatography | nLC-ESI-MS/MS | 19 (5%) | 600–42,000 | Phenotypic cell wall assay using knock-outs | 3 |
| Plasma membrane | FY1679 (n), YPD, 28 °C | Direct mechanical disruption | 1) Acid precipitation, 2) sucrose density gradient (34–56%) | 0.4% deoxycholate | SDS PAGE | CTAB/SDS PAGE | MALDI-MS/MS | 48 (19%) | 377–1,255,722 | 5 | |
| Plasma membrane | FY1679 (n), YPD | Direct mechanical disruption | 1) Acid precipitation, 2) Sucrose density gradient (43–53%) | EDTA, n-octyl glucoside | ATPase assays, SDS-PAGE | Anion EC/LDS-PAGE | PMF: MALDI-MS, ESI-MS/MS | 86 (21%) | 166–1,255,722 | 4 |
a (n) or (2n) after the strain name indicates haploid or diploid strain, respectively.
b Unless indicated otherwise, the cells were grown aerobically at 30 °C in YPD medium containing 1% yeast extract, 2% Bacto peptone, 2% glucose.
c YPL medium contained 1% yeast extract, 2% Bacto peptone, 0.5% lactic acid.
d YPG medium contained 1% yeast extract, 2% Bacto peptone, 3% glycerol, pH 5.0.
e Digestion was carried out using four proteases: trypsin, chymotrypsin, Glu-C, and subtilisin.
f SCL and SCD media are complete synthetic media supplemented with lactic acid or glucose, respectively.
g YNO contained 0.1% yeast extract, 0.5% ammonium sulfate, 1.7 g/liter yeast nitrogen base, 0.02% Tween 40, 0.3% glucose, 0.1% oleic acid.
h SCIM contained 0.5% yeast extract, 0.1% peptone, 0.79 g/liter complete synthetic medium mixture, 0.5% ammonium sulfate, 1.7 g/liter yeast nitrogen base, 0.1% Tween 40, 0.1% glucose, 0.15% oleic acid.