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PeptideAtlas provides a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments collected for human, mouse, yeast, and several other organisms.
(last updated: Nov 28, 2017)
Data or Model Molecule| Accession | Sample Title | Summary | Organism | Characteristics | Instrument | Treatment | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| PAe000337 | human serum fractionation/depletion methods, 21_unfract | 21_unfract | Human | Male, Serum, Sigma-Aldrich, catalog #H4522, lot #043K0502 | LTQ | Ammonium bicarbonate (50 mM) was added to achieve a final methanol concentration of 20% and the samples were digested with Trypsin Gold (Promega, Madison, WI) at a protein to enzyme ratio of 50:1 (w/w) at 37°C for 6 h. The samples were dried in a SpeedVac and resuspended in 50 mM ammonium bicarbonate prior to LC-MS analysis. | Human serum samples were denatured and reduced with 60% methanol and 10 mM dithiothreitol (DTT) at 60°C for 1 h and alkylated with 50 mM iodoacetamide (IAM) at room temperature in the dark for 30 min. | Unfractionated | PMID:17269739 | ||
| PAe000324 | yeast_quality_control | yeast_quality_control Yeast cell lysate was run on LTQ, LTQ_FT and QTOF. | Yeast | BY4741 (MATa, leu2D0, met15D0, ura3D0, his3D1) | LTQ | Yeast grown in synthetic complete medium at 30°C to log phase, harvested and diluted into 30mL synthetic complete media to a cell count of 3 × 10⁶/mL. | A 150µL portion of reduced yeast protein lysate was alkylated in 25mM of iodoacetamide and diluted to 1M urea with 50mM ammonium bicarbonate. 12µg of Trypsin Gold (Promega, Madison, WI) was added to the reduced and alkylated yeast protein lysate and trypsin digest was carried out overnight at 37°C. Trypsin was inactivated by addition of glacial acetic acid and insoluble material was removed by centrifugation. Peptides were purified using 3 mL SPEC C18 columns and concentrated in a SpeedVac. | After 4 h of growth at 30°C cells were harvested and washed three times with ice cold dH₂O. Cells were lysed by incubation with 1mL of ice cold 10% TCA for 1 h at 4°C. Protein precipitates were collected by centrifugation, washed twice with 1 mL cold 90% acetone, and dried in a SpeedVac (Thermo Savant, Holbrook, NY). Proteins were solubilized in 300µL of 8 M urea, 50mM ammonium bicarbonate and reduced by incubation at 56°C for 1 h in the presence of 15 mM DTT. | Unfractionated. | PMID:16823959 | |
| PAe000032 | Human bronchoalveolar lavage fluid, 031003_BALF2 | Human bronchoalveolar lavage fluid Straight tryptic digest of 2mg equivalent of human bronchoalveolar lavage fluid (BALF) sample 2. Trypsinized sample was cleaned up/fractionated over 2.1 x 200 mm PolysulfoethylA (SCX) column, collecting 1 minute (= 200 ul) fractions. 11 fractions were processed separately over RP-LC-MS/MS using DECA quad-IT MS. Samples were subject to a 165 minute RP gradient (5%-35% ACN) and up to 4 MS/MS scans were allowable per survey scan. | Human | Bronchoalveolar lavage fluid | LCQ DECA | Not applicable | Not applicable | BALF proteins were concentrated by ice-cold acetone precipitation. BALF containing 2 mg of protein underwent digestion with trypsin (20µg, sequencing grade; Promega, Madison, WI) overnight at 37°C to allow complete digestion. | Bronchoalveolar lavage (BAL) was performed as previously described in papers. (Matute-Bello G, et al., Am J Respir Crit Care Med, 1997, 156:1969–1977; Steinberg KP et al., Am J Respir Crit Care Med 1994, 150:113–122; Greene KE, et al., Am J Respir Crit Care Med 1999, 160:1843–1850) Briefly, five separate 30-ml aliquots of 0.89% sterile saline were instilled into the right middle lobe or lingula. The BAL recovery averaged 75 ml (49% return) and was not statistically different between the different ARDS groups by one-way analysis of variance (P > 0.05). BALF was centrifuged immediately after collection, and cell-free supernatants were aliquoted into polypropylene tubes and stored at 70°C. Total protein measurements were made on aliquots of supernatants using a modified Lowry method. | To prepare for strong-cation exchange chromatography and to reduce the salt concentration, the digested resulting peptide solutions were diluted eightfold with running buffer (5 mmol/L KH₂PO₄, 25% acetonitrile, pH 3), and their pH was reduced to 2.9 with phosphoric acid (H₃PO₄). The peptide solutions were passed over a 2.1×200 mm, 5-µm particle, 300-Å pore Polysulfoethyl A column (PolyLC; Columbia, MD), washed with running buffer, and then eluted with a 50-minute biphasic gradient of 0 to 25% elution buffer (running buffer plus 350 mmol/L potassium chloride) in 0 to 30 minutes followed by 25 to 100% elution buffer in 30 to 50 minutes. Flow rate was constant at 0.2 ml/minute. Sixteen 2-minute (0.4-ml) fractions were collected. Fractions from strong-cation exchange chromatography were completely dried down in a Speed-Vac (Thermo-Savant, Milford, MA) and redissolved in 0.1% trifluoroacetic acid. To desalt, fractions were loaded onto Oasis mixed-mode cation-exchange cartridges (Waters, Milford, MA), washed with 0.1% trifluoroacetic acid, and eluted with 0.1% trifluoroacetic acid, 80% acetonitrile solution. The samples were again dried down and redissolved in 0.2% acetic acid and transferred to autosampler vials for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. | PMID:16816363 |
| PAe000033 | Human bronchoalveolar lavage fluid, 032403_BALF4_glypep | Human bronchoalveolar lavage fluid Two different amounts of bronchoalveolar lavage fluid (BALF) sample 4 were subject to Hui Zhang's n-linked specific glycopeptide purification method and then processed separately over RP-LC-MS/MS using DECA quad-IT MS. Sample '970' was derived from 970 ul of BALF 4, containing an estimated 1,125 ug of protein Sample '1940' was derived from 1,940 ul of BALF 4, containing an estimated 2,250 ug of protein. After glycopeptide purification samples were subject to a 165 minute RP gradient (5%-35% ACN) and up to 4 MS/MS scans were allowable per survey scan. | Human | Bronchoalveolar lavage fluid | LCQ DECA | Not applicable | Not applicable | BALF proteins were concentrated by ice-cold acetone precipitation. BALF containing 2 mg of protein underwent digestion with trypsin (20µg, sequencing grade; Promega, Madison, WI) overnight at 37°C to allow complete digestion. | Bronchoalveolar lavage (BAL) was performed as previously described in papers. (Matute-Bello G, et al., Am J Respir Crit Care Med, 1997, 156:1969–1977; Steinberg KP et al., Am J Respir Crit Care Med 1994, 150:113–122; Greene KE, et al., Am J Respir Crit Care Med 1999, 160:1843–1850) Briefly, five separate 30-ml aliquots of 0.89% sterile saline were instilled into the right middle lobe or lingula. The BAL recovery averaged 75 ml (49% return) and was not statistically different between the different ARDS groups by one-way analysis of variance (P > 0.05). BALF was centrifuged immediately after collection, and cell-free supernatants were aliquoted into polypropylene tubes and stored at 70°C. Total protein measurements were made on aliquots of supernatants using a modified Lowry method. | To prepare for strong-cation exchange chromatography and to reduce the salt concentration, the digested resulting peptide solutions were diluted eightfold with running buffer (5 mmol/L KH₂PO₄, 25% acetonitrile, pH 3), and their pH was reduced to 2.9 with phosphoric acid (H₃PO₄). The peptide solutions were passed over a 2.1×200 mm, 5-µm particle, 300-Å pore Polysulfoethyl A column (PolyLC; Columbia, MD), washed with running buffer, and then eluted with a 50-minute biphasic gradient of 0 to 25% elution buffer (running buffer plus 350 mmol/L potassium chloride) in 0 to 30 minutes followed by 25 to 100% elution buffer in 30 to 50 minutes. Flow rate was constant at 0.2 ml/minute. Sixteen 2-minute (0.4-ml) fractions were collected. Fractions from strong-cation exchange chromatography were completely dried down in a Speed-Vac (Thermo-Savant, Milford, MA) and redissolved in 0.1% trifluoroacetic acid. To desalt, fractions were loaded onto Oasis mixed-mode cation-exchange cartridges (Waters, Milford, MA), washed with 0.1% trifluoroacetic acid, and eluted with 0.1% trifluoroacetic acid, 80% acetonitrile solution. The samples were again dried down and redissolved in 0.2% acetic acid and transferred to autosampler vials for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. | PMID:16816363 |
| PAe000335 | human serum fractionation/depletion methods, 18_size | 18_size | Human | Male, Serum, Sigma-Aldrich, catalog #H4522, lot #043K0502 | LTQ | Ammonium bicarbonate (50 mM) was added to achieve a final methanol concentration of 20% and the samples were digested with Trypsin Gold (Promega, Madison, WI) at a protein to enzyme ratio of 50:1 (w/w) at 37°C for 6 h. The samples were dried in a SpeedVac and resuspended in 50 mM ammonium bicarbonate prior to LC-MS analysis. | Human serum samples were denatured and reduced with 60% methanol and 10 mM dithiothreitol (DTT) at 60°C for 1 h and alkylated with 50 mM iodoacetamide (IAM) at room temperature in the dark for 30 min. | Size fractionation Human serum samples were diluted 1:5 in 50 mM ammonium bicarbonate/20% ACN (pH 8.0) and centrifuged at 16 000×g for 5 min at 4°C to pellet any precipitate. A Millipore filter unit with 30 kDa molecular weight cutoff was rinsed with 50 mM ammonium bicarbonate/20% ACN and centrifuged at 2000×g for 5 min. Diluted serum sample (400µL) was added to the washed filter unit and centrifuged at 5000×g at 4°C for 30 min. The flow-through aliquot was transferred to a siliconized sterile vial. Ammonium bicarbonate/20% ACN (200µL) was added to the remaining sample and centrifuged in the same way as in the previous step. This cycle was repeated three times. Coomassie protein assay (Pierce, Rockford, IL) was performed on the pooled flowthrough aliquot to verify protein concentration. The flow through aliquot was dried in a SpeedVac and stored at -80°C until analysis. | PMID:17269739 | ||
| PAe000026 | HeLa nuclear extracts, TREX binding factor assay, ICAT | For identification of the TREX binding factor, two samples were prepared by DNA affinity chromatography using HeLa nuclear extracts. The first sample was purified using a TREX binding site column. The second sample was prepared using a mutated TREX binding site column. Proteins were differentially labeled with old ICAT reagents , combined, prepared for mass spectrometry, and analyzed on an ion trap mass spectrometer. see project | LCQ DECA | HeLa cells adapted for growth in suspension (HeLa S) were grown in RPMI medium supplemented with 10% fetal bovine serum and antibiotics (100 U of penicillin/ml and 0.1mg of streptomycin/ml) in 500 ml spinner flasks to a density of ~10⁶ cells/ml. Approximately 5 × 10⁸ cells were harvested every 3 to 4 days for nuclear extraction, and nuclear extracts were frozen at -70°C for ~5 weeks, until a total of 5 × 10⁹ cells were obtained. Mouse MM14 skeletal myoblasts were grown on 100mm collagen-coated tissue culture dishes in proliferation medium (Ham’s F-10C supplemented with 15% horse serum and 2ng of basic fibroblast growth factor/ml). Myogenic differentiation was induced by switching to differentiation medium (Ham’s F-10C supplemented with 1.5% horse serum and 1 µM insulin). Cells were maintained for 48 h in differentiation medium prior to harvesting. These cultures contained > 90% terminally differentiated myocytes as assessed by immunostaining a parallel culture with the myosin-specific antibody MF-20. Ventricular myocardiocytes from 2- to 3-day-old Sprague-Dawley rats were isolated and cultured as described in Nguyen et al., Transgenic Res. 12: 337–349. These cultures contained > 90% myocardiocytes as assessed by immunohistochemistry with the MF-20 antibody. | Proteins eluted from hTrex and hTrex-mt beads were diluted threefold with 20 mM Tris-HCl (pH 8.3), 1 mM EDTA to bring the salt concentration to 0.1 M and then concentrated in Microcon 3 devices (Amicon). Proteins were denatured by adding sodium dodecyl sulfate (SDS) to 0.3% and heating for 5 min at 100°C and then reduced with 5 mM tri-n-butylphosphine for 30 min at 37°C. Samples were diluted sixfold with 20 mM Tris-HCl (pH 8.3), 1 mM EDTA, and 7.2 M urea. Isotopically heavy and normal isotope-coded affinity tag (ICAT) reagents were added to hTrex and hTrex-mt samples, respectively, to 1.75 mM, and samples were incubated for 90 min at 22°C. Labeling reactions were quenched by adding dithiothreitol to 10 mM for 15 min at 37°C. Samples were combined and digested for 2 h at 37°C with 10 µg of endoproteinase Lys-C (Boehringer-Mannheim), which cleaves on the carboxy-terminal side of lysine residues. SDS and urea concentrations were reduced to 0.02% and 1.2 M, respectively, by adding 20 mM Tris-HCl (pH 8.3) and 1 mM EDTA, and the sample was digested overnight at 37°C with trypsin (sequencing grade modified; 1 : 20 [wt/wt]; Promega), which cleaves on the carboxy-terminal side of arginine and lysine residues. | Crude nuclear extracts from cultured cells and embryonic tissues were prepared as described in Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983, Nucleic Acids Res. 11: 1475–1489. Embryonic tissues were minced on ice before the release of nuclei. Crude nuclear extracts from adult mouse ventricle were prepared as Zahradka, P., D. E. Larson, and B. H. Sells. 1989, Cell Res. 185: 8–20. All nuclear extracts contained a cocktail of several protease inhibitors (product no.P8340; Sigma). | The sample was diluted to 15 ml with buffer A (5 mM KH₂PO₄ [pH 3], 25% CH₃CN), and the pH was adjusted to 3 with 10% trifluoroacetic acid. Peptides were fractionated by SCX high-performance liquid chromatography (2.1 by 200 mm PolySULFOETHYL A [PolyLC]) by running the following gradient: 0 to 15% buffer B (5mM KH₂PO₄[pH 3], 600 mM KCl, 25% CH₃CN) over 30 min, 15 to 60% buffer B in 20 min, and 60 to 100% buffer B in 15 min at 0.2 ml/min. A total of 53 fractions were collected. ICAT-labeled peptides were purified from unlabeled peptides by passing each fraction over monomeric avidin cartridges (ABI) and washing with 2 × PBS (pH 7.2), 1 × PBS (pH 7.2), and 50 mM NH₄HCO₃ (pH 8.3), 20% CH₃OH. Peptides were eluted with 0.4% trifluoroacetic acid in 30% CH₃CN and dried under reduced pressure, and each fraction was resuspended in 12 µl of 0.2% acetic acid. | PMID:14966291 | |||
| PAe000084 | Saccharomyces cerevisiae (yeast) treated with mating hormone, ICAT | Catalogue changes in budding yeast (S. cerevisiae) protein expression following brief treatment with mating hormone, with the endpoint of identifying translationally controlled proteins. baseline (light ICAT label) is protein from an asynchrnous culture same harvest and chromatographic methods as with TSAA timepoint experiments, but in this case measurements taken 30 minutes after alpha-factor application (no alpha-factor removal); baseline (light ICAT label) is protein from an asynchrnous culture | Yeast | BY1782 (MATa cdc15-2 tyr1 leu2 ura3 his7 gal1), BY2125 (MATa ade2-1 his3-11 leu2-3, 112 trp1-1 ura3 can1-100 ssdl-d) | LTQ | Cultures were grown at 25°C in a shaking incubator except during the cdc15 arrest (37°C) and growth of cells for the β-galactosidase assays (30°C). cdc15 Synchronization -- Cells were grown in 400 ml of YEPD medium (1% yeast extract, 2% peptone, 2% dextrose) to a density of 5–8×10⁶cells/ml, and then the cultures were shifted to 37°C for 3 h. To release cells from arrest, flasks were swirled rapidly in an ice-water bath so that the culture temperature reached 25°C in less than 1 min. Two separate experiments were conducted: one in which samples for probe preparation were taken every 10 min from 0 to 200min and one in which samples were taken every 20 min from 0 to 60min. The collection of samples took place over several months in four sets (0–50 min, 60–100 min, 110–150 min, and 160–200 min). At the same time that samples for TSAA were taken, aliquots of cells were also removed for bud counts and total RNA isolation. α-Factor Synchronization—Cells were grown to a density of 5–8×10⁶cells/ml at which point α-factor was added. The cells were fully arrested after 2 h. To release cells from arrest they were centrifuged for 2 min at 3000×g and resuspended in 400 ml of fresh medium. Two separate experiments were conducted: one in which samples were taken every 10 min for 0 to 190 min and one in which samples were taken every 10 min from 0 to 50 min. For each time point, RNA isolated from three separate cultures was pooled before labeling. At the same time that samples for TSAA were taken, aliquots of cells were removed for bud counts and total RNA isolation. | Trypsin | All steps took place in a 4°C cold room. To prepare polysomes, ~100 ml of culture was poured into a flask containing ~75 ml of crushed frozen YEPD. The flask was swirled rapidly on ice for 1–2 min and then centrifuged for 2 min at 3000 × g to pellet the cells. The pellet was resuspended in 10 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 100mM NaCl, 30mM MgCl₂, ] 500µg/ml heparin, 50µg/ml cycloheximide) and centrifuged a second time. The pellet was resuspended in 1 ml of lysis buffer and transferred to a 15-ml conical tube containing 2 g of glass beads (~500-µm diameter, Sigma). The tubes were vortexed eight times for 30 s each. Tubes were placed on ice for 30 s between rounds of vortexing. Tubes were centrifuged for 1 min in a clinical centrifuge to pellet the beads, and the supernatant was removed. Lysis buffer (0.5 ml) was added to the beads, the beads were vortexed for 15 s, and the tube was centrifuged again to pellet the beads. The supernatant was removed and added to the first supernatant. The pooled supernatants were centrifuged in a microcentrifuge at top speed (20,000 × g) for 1 min to pellet debris, and the supernatant was transferred to a fresh tube. The A₂₆₀ of the supernatant was measured. For the cdc15 experiments 50–60 absorbance units were loaded per gradient. For the α-factor experiments, 25 absorbance units were loaded per gradient. | The samples were loaded on a 10-ml linear gradient of 7–47% sucrose with a 1-ml 2 M sucrose cushion at the bottom and centrifuged for 2 h in an SW40 Ti swinging bucket rotor at 4°C and 39,000 rpm. The gradient buffer consisted of 50 mM Tris acetate, pH 7.0, 50 mM NH₄Cl, 12 mM MgCl₂, and 1 mM dithiothreitol. After centrifugation, the gradients were collected into 24 0.5-ml fractions. Fifty microliters of 3 M NaOAc, pH 5.2, and 50 µl of 10% SDS were added to each fraction. During fractionation a running trace of A₂₅₄ was recorded to monitor the polysome profile. | PMID:12684541 | |
| PAe000116 | Human Erythroleukemia K562 cell line,Q | Human huamn erythroleukemia K5632 cell line I think... | Human | K562 (erythroleukemia) | LTQ | K562 grown in suspension. | Proteins in the pooled fractions were alkylated with iodoacetamide, desalted into 100 mM NH₄HCO₃, digested, lyophilized, and processed in the same manner as sample 1 (referring PSM1018) and sample 2 (referring PSM1019). | Cells were washed twice by centrifugation, and pellets were flash frozen in liquid nitrogen. Cell pellets were suspended in lysis buffer (140mM potassium phosphate, pH 7.4, 150mM NaF, 1mM Na₃VO₄, 6mM EDTA, 6mM EGTA, 250mM NaCl, 4mM DTT) containing 40µg/mL leupeptin, 5µg/mL pepstatin A, 4mM benzamidine, and 20mM PMSF and sonicated 4 × 15 s at 4°C (Branson, microtip probe). Lysates were centrifuged at 200000g for 30 min at 4°C and soluble proteins recovered in the supernatant. Typically 10⁸ cells yielded ~15mg of protein. | Proteins were homogenized in lysis buffer on a gel filtration column (Sephacryl HR300 (26/60), Amersham) equilibrated in lysis buffer. The column was run at 1.3 mL/min, collecting 8-mL fractions over the included volume. | PMID:15228325 | |
| PAe000333 | human serum fractionation/depletion methods, 19_proteinag | 19_proteinag | Human | Male, Serum, Sigma-Aldrich, catalog #H4522, lot #043K0502 | LTQ | Ammonium bicarbonate (50 mM) was added to achieve a final methanol concentration of 20% and the samples were digested with Trypsin Gold (Promega, Madison, WI) at a protein to enzyme ratio of 50:1 (w/w) at 37°C for 6 h. The samples were dried in a SpeedVac and resuspended in 50 mM ammonium bicarbonate prior to LC-MS analysis. | Human serum samples were denatured and reduced with 60% methanol and 10 mM dithiothreitol (DTT) at 60°C for 1 h and alkylated with 50 mM iodoacetamide (IAM) at room temperature in the dark for 30 min. | Protein A/G depletion Immunoglobulins were depleted by using a 2 mL pre-packed immobilized Protein A/G column (Pierce, Rockford, IL) per the manufacturer's instructions. Briefly, 50 mL of human serum sample was diluted 1:5 with binding buffer and centrifuged at 10 000×g for 20 min. The supernatant was applied to a Protein A/G column that was preequilibrated with 5 mL of binding buffer. 15 mL of binding buffer was added to wash the column, and the flow-through fraction was collected. Sample was then dialyzed against 50 mM ammonium bicarbonate and lyophilized. | PMID:17269739 | ||
| PAe000007 | Human prostate cancer cell lines, nuclear, ICAT | Human prostate cancer cell lines: LNCaP, CL-1, nuclear. Labeled with old ICAT. nuclear fraction comparison with ICAT | Human | Human prostate cancer cell lines LNCaP, CL-1. | LTQ | LNCaP and CL1 cells were grown as described by Tso et al. Cancer J Sci Am 2000, 6: 220–33. | Procedures based on Han et al., Nat Biotechnol 2001; 19: 946–51. | Fractionation of cells into cytosolic, microsomal, and nuclear fractions according to Han et al., Nat Biotechnol 2001; 19: 946–51. | Procedures based on Han et al., Nat Biotechnol 2001; 19: 946–51. | PMID:15833837 | |
| PAe000006 | Human prostate cancer cell lines, microsomal, ICAT | Human prostate cancer cell lines: LNCaP, CL-1, microsomal. Labeled with old ICAT. microsomal comparison with ICAT | Human | Human prostate cancer cell lines LNCaP, CL-1. | LCQ DECA | LNCaP and CL1 cells were grown as described by Tso et al. Cancer J Sci Am 2000, 6: 220–33. | Procedures based on Han et al., Nat Biotechnol 2001; 19: 946–51. | Fractionation of cells into cytosolic, microsomal, and nuclear fractions according to Han et al., Nat Biotechnol 2001; 19: 946–51. | Procedures based on Han et al., Nat Biotechnol 2001; 19: 946–51. | PMID:15833837 | |
| PAe000005 | Human prostate cancer cell lines, cytosolic, ICAT | Human prostate cancer cell lines: LNCaP, CL-1, cytosolic. labeled with old ICAT cytosolic fraction comparison with ICAT | Human | Human prostate cancer cell lines LNCaP, CL-1. | LCQ DECA | LNCaP and CL1 cells were grown as described by Tso et al. Cancer J Sci Am 2000, 6: 220–33. | Procedures based on Han et al., Nat Biotechnol 2001; 19: 946–51. | Fractionation of cells into cytosolic, microsomal, and nuclear fractions according to Han et al., Nat Biotechnol 2001; 19: 946–51. | Procedures based on Han et al., Nat Biotechnol 2001; 19: 946–51. | PMID:15833837 | |
| PAe000014 | Human Caco-2 epithelial cell line, ICAT | Human Caco-2 intestinal epithelial cell line; labeled with CL-ICAT; IPI Using Caco-2 cells, a human intestinal epithelial cell line. Goal is to determine the intestinal response to infection by enteropathogenic E. coli. Typical experiments involve 6e4 cells, infected with pathogenic E. coli at a MOI of ca. 100:1. Experiments are done in 6-well plates. After washing the cells with PBS to remove bacteria, cells are lysed in 1% Triton in PBS. After clearing the extract with a 5' 12000g spin, acetone precipitation is performed to recover the total protein. The precipitate is then air-dried and resuspended in 6M urea + 0.1% SDS. Each sample (control vs. infected) is then subject to clICAT labeling (control = d0; infected = d9) followed by cation exchange cleanup/fractionation and subsequent avidin purification. Some fractions are then combined and all are then cleaned up with sep-pak C18 (1 cc, Waters) to wash away salt. The sep-pak eluent was then dried down and the pellet was redissolved in 8 ul 0.2% acetic acid. 2 ul thereof was processed over RP-LC-MS/MS using DECA quad-IT MS. Samples were subject to a 140 minute RP gradient (10%-35% ACN) and up to 4 MS/MS scans were allowable per survey scan. | Human | Caco-2, Wild type and type III secretion system( TTSS)-deficient enteropathogenic Escherichia coli | LCQ DECA | Bacterial cultures (wild-type Enteropathogenic Escherichia coli (EPEC) E2348/69 and EPEC E2348/69∆escN, N-) grown overnight were subcultured 1:50 into Dulbecco's modified Eagle's medium and grown for 3 h at 37°C, 5% CO₂, without shaking. Bacteria were applied to Caco-2 cells for 4 h at a multiplicity of infection of 50:1. Following infection, the media was aspirated and cells were washed 8 times with PBS. | Human Caco-2 cells were grown at 37°C, 5% CO₂, in Dulbecco's modified Eagle's medium supplemented with 10% decomplemented fetal calf serum, and 1% non-essential amino acids. Cells were cultured in 24-mm diameter polyester Transwell plates (Costar) for at least 21 days prior to infection. | Trypsin was added at 1:50 (w/w) and digested O/N at 37°C. The peptide solution was passed over a PolysulfoethylA" column and an avidin cartridge, followed by cleavage of the biotin portion of the ICAT" and µLC-MS and µLC-MS/MS analysis. | Cells were lysed in 500 µl of 1% Triton X-100 in PBS and centrifuged at 12,000 x g for 5 min. The supernatant was transferred to 5 volumes of cold acetone, precipitated, and resuspended in 0.1% SDS, 6 M urea. Caco-2 cell lysates (1.1 mg; infected with either wt or N-) were solubilized in 300 µL labelling buffer (0.05% SDS, 50 mM Tris pH 8.3, 5 mM EDTA, 6 M urea). Proteins were reduced with 5 mM TCEP (Tris(2-carboxyethyl)phosphine) for 30 min at 37°C. 3.9 molar equivalents of ICAT reagent [wt=d₀(light); N-=d₉(heavy)] per cysteine equivalent were added in the dark, for 2 h, at 37°C. Samples were quenched with a 10-fold molar excess of DTT, for 5 min, at 22°C, mixed, and diluted to a final [urea] of 1 M. | The peptide solution was passed over a PolysulfoethylA" column (PolyLC, Columbia, MD). The column was washed with solvent A (5 mM monobasic potassium phosphate, 25% acetonitrile, pH 3) and the peptides were then eluted with a 50 min biphasic gradient of 0-25% solvent B (solvent A + 600 mM potassium chloride) in 0-30 min followed by 25-100% solvent B in 30-50 min. Flow rate was constant at 0.44 ml/min. 0.44 ml eluent fractions were collected each min and neutralized with 44 µl of sodium phosphate, pH 10.0, and subsequently passed over an avidin cartridge (Applied Biosystems, Framingham, MA), and washed with 2X phosphate-buffered saline, water, and 50 mM ammonium bicarbonate in 20% methanol pH 8.3. Biotinylated peptides were eluted from the avidin with a 30% acetonitrile, 0.4% trifluoroacetic acid solution and then dried down. All of the preceding chromatography steps were performed automatically on a Vision" multidimensional high-performance liquid chromatography machine (Applied Biosystems). The sample pellets were dissolved in cleavage buffer (Applied Biosystems) to cleave away the biotin portion of the ICAT" tag. The samples were again dried down and then redissolved in 0.2% acetic acid and transferred to autosampler vials for MS analysis. | PMID:14988394 |
| PAe000168 | Qian Plasma non alkylated samples | Qian Plasma non alkylated samples Weijun Qian's paper (Qian et. al., Molecular and Cellular Proteomics 2005, 4, 700-709) was compiled from 461 data files, totalling 9.0 GB. The files from the Qian paper are mainly global, trypsinized plasma. The WQ-PLAS files were all analyzed with static iodoacetimide on C while the remaining files (plasma12, plasmaJK, & plasmaJon) were not alkylated and therefore were not analyzed with any modifications enabled. | Human | Adult, Human plasma | LCQ DECA XP | Aliquots of 200µl each of the control and LPS-treated plasma samples were diluted and denatured using 8M urea, 50mM NH₄HCO₃, pH 8.2 for 1 h at 37°C and reduced with 10mM DTT for 30 min at 37°C. Protein cysteinyl residues were alkylated with 40mM iodoacetamide for 90 min at room temperature, and samples were desalted using a prepacked PD-10 column containing Sephadex G-25 (Amersham Biosciences). The protein concentrations for the desalted samples were measured using a BCA protein assay (Pierce) that gave total protein amounts of 15.0 and 13.9mg for the control and LPS-treated plasma samples, respectively. | The samples were digested into peptides using sequencing grade trypsin (Promega, Madison, WI) overnight at 37°C with a 1:50 (w/w) trypsin-to-protein ratio. Tryptic activity of residual trypsin was quenched by boiling the samples for 10 min and immediately placing the samples on ice. | The ¹⁶O/¹⁸O-labeled peptide samples from the control and LPS-treated plasma samples were fractionated by Strong Cation Exchange (SCX) Fractionation. The lyophilized sample was resuspended in 1.5ml of 10mM ammonium formate, 25% acetonitrile, pH 3.0 and injected onto a 10 × 4.6-mm guard column attached to a polysulfoethyl A 200 × 4.6-mm (5-µm, 300-Å) column (Poly LC, Columbia, MD). The mobile phases consisted of solvent A (10mM ammonium formate, 25% acetonitrile, pH 3.0) and solvent B (500mM ammonium formate, 25% acetonitrile, pH 6.8). After sample loading, the separation was isocratic for 10 min with 100% solvent A with a flow rate of 1ml/min. Peptides were eluted using sequential linear gradients from 100% solvent A to 50% solvent B over 40 min and from 50% solvent B to 100% solvent B over another 10 min. The mobile phase was held at 100% solvent B for another 15 min. 1-ml fractions (1 min/fraction) were collected after the start of the gradient using a Shimadzu FRC-10A fraction collector (Kyoto, Japan) and combined into 30 fractions. Each fraction was lyophilized and analyzed by reversed-phase LC-FTICR. | PMID:15753121 | ||
| PAe000169 | Qian Plasma alkylated samples | Qian Plasma alkylated samples Weijun Qian's paper (Qian et. al., Molecular and Cellular Proteomics 2005, 4, 700-709) was compiled from 461 data files, totalling 9.0 GB. The files from the Qian paper are mainly global, trypsinized plasma. The WQ-PLAS files were all analyzed with static iodoacetimide on C while the remaining files (plasma12, plasmaJK, & plasmaJon) were not alkylated and therefore were not analyzed with any modifications enabled. | Human | Adult, Human plasma | LCQ Duo | Aliquots of 200µl each of the control and LPS-treated plasma samples were diluted and denatured using 8M urea, 50mM NH₄HCO₃, pH 8.2 for 1 h at 37°C and reduced with 10mM DTT for 30 min at 37°C. Protein cysteinyl residues were alkylated with 40mM iodoacetamide for 90 min at room temperature, and samples were desalted using a prepacked PD-10 column containing Sephadex G-25 (Amersham Biosciences). The protein concentrations for the desalted samples were measured using a BCA protein assay (Pierce) that gave total protein amounts of 15.0 and 13.9mg for the control and LPS-treated plasma samples, respectively. | The samples were digested into peptides using sequencing grade trypsin (Promega, Madison, WI) overnight at 37°C with a 1:50 (w/w) trypsin-to-protein ratio. Tryptic activity of residual trypsin was quenched by boiling the samples for 10 min and immediately placing the samples on ice. | The ¹⁶O/¹⁸O-labeled peptide samples from the control and LPS-treated plasma samples were fractionated by Strong Cation Exchange (SCX) Fractionation. The lyophilized sample was resuspended in 1.5ml of 10mM ammonium formate, 25% acetonitrile, pH 3.0 and injected onto a 10 × 4.6-mm guard column attached to a polysulfoethyl A 200 × 4.6-mm (5-µm, 300-Å) column (Poly LC, Columbia, MD). The mobile phases consisted of solvent A (10mM ammonium formate, 25% acetonitrile, pH 3.0) and solvent B (500mM ammonium formate, 25% acetonitrile, pH 6.8). After sample loading, the separation was isocratic for 10 min with 100% solvent A with a flow rate of 1ml/min. Peptides were eluted using sequential linear gradients from 100% solvent A to 50% solvent B over 40 min and from 50% solvent B to 100% solvent B over another 10 min. The mobile phase was held at 100% solvent B for another 15 min. 1-ml fractions (1 min/fraction) were collected after the start of the gradient using a Shimadzu FRC-10A fraction collector (Kyoto, Japan) and combined into 30 fractions. Each fraction was lyophilized and analyzed by reversed-phase LC-FTICR. | PMID:15753121 | ||
| PAe000276 | Qian Plasma alkylated samples | Qian Plasma alkylated samples Weijun Qian's paper (Qian et. al., Molecular and Cellular Proteomics 2005, 4, 700-709) was compiled from 461 data files, totalling 9.0 GB. The files from the Qian paper are mainly global, trypsinized plasma. The WQ-PLAS files were all analyzed with static iodoacetimide on C while the remaining files (plasma12, plasmaJK, & plasmaJon) were not alkylated and therefore were not analyzed with any modifications enabled. | Human | Adult, Human plasma | LCQ Duo | Aliquots of 200µl each of the control and LPS-treated plasma samples were diluted and denatured using 8M urea, 50mM NH₄HCO₃, pH 8.2 for 1 h at 37°C and reduced with 10mM DTT for 30 min at 37°C. Protein cysteinyl residues were alkylated with 40mM iodoacetamide for 90 min at room temperature, and samples were desalted using a prepacked PD-10 column containing Sephadex G-25 (Amersham Biosciences). The protein concentrations for the desalted samples were measured using a BCA protein assay (Pierce) that gave total protein amounts of 15.0 and 13.9mg for the control and LPS-treated plasma samples, respectively. | The samples were digested into peptides using sequencing grade trypsin (Promega, Madison, WI) overnight at 37°C with a 1:50 (w/w) trypsin-to-protein ratio. Tryptic activity of residual trypsin was quenched by boiling the samples for 10 min and immediately placing the samples on ice. | The ¹⁶O/¹⁸O-labeled peptide samples from the control and LPS-treated plasma samples were fractionated by Strong Cation Exchange (SCX) Fractionation. The lyophilized sample was resuspended in 1.5ml of 10mM ammonium formate, 25% acetonitrile, pH 3.0 and injected onto a 10 × 4.6-mm guard column attached to a polysulfoethyl A 200 × 4.6-mm (5-µm, 300-Å) column (Poly LC, Columbia, MD). The mobile phases consisted of solvent A (10mM ammonium formate, 25% acetonitrile, pH 3.0) and solvent B (500mM ammonium formate, 25% acetonitrile, pH 6.8). After sample loading, the separation was isocratic for 10 min with 100% solvent A with a flow rate of 1ml/min. Peptides were eluted using sequential linear gradients from 100% solvent A to 50% solvent B over 40 min and from 50% solvent B to 100% solvent B over another 10 min. The mobile phase was held at 100% solvent B for another 15 min. 1-ml fractions (1 min/fraction) were collected after the start of the gradient using a Shimadzu FRC-10A fraction collector (Kyoto, Japan) and combined into 30 fractions. Each fraction was lyophilized and analyzed by reversed-phase LC-FTICR. | |||
| PAe000278 | Qian Plasma non alkylated samples | Qian Plasma non alkylated samples Weijun Qian's paper (Qian et. al., Molecular and Cellular Proteomics 2005, 4, 700-709) was compiled from 461 data files, totalling 9.0 GB. The files from the Qian paper are mainly global, trypsinized plasma. The WQ-PLAS files were all analyzed with static iodoacetimide on C while the remaining files (plasma12, plasmaJK, & plasmaJon) were not alkylated and therefore were not analyzed with any modifications enabled. | Human | Adult, Human plasma | LCQ DECA XP | Aliquots of 200µl each of the control and LPS-treated plasma samples were diluted and denatured using 8M urea, 50mM NH₄HCO₃, pH 8.2 for 1 h at 37°C and reduced with 10mM DTT for 30 min at 37°C. Protein cysteinyl residues were alkylated with 40mM iodoacetamide for 90 min at room temperature, and samples were desalted using a prepacked PD-10 column containing Sephadex G-25 (Amersham Biosciences). The protein concentrations for the desalted samples were measured using a BCA protein assay (Pierce) that gave total protein amounts of 15.0 and 13.9mg for the control and LPS-treated plasma samples, respectively. | The samples were digested into peptides using sequencing grade trypsin (Promega, Madison, WI) overnight at 37°C with a 1:50 (w/w) trypsin-to-protein ratio. Tryptic activity of residual trypsin was quenched by boiling the samples for 10 min and immediately placing the samples on ice. | The ¹⁶O/¹⁸O-labeled peptide samples from the control and LPS-treated plasma samples were fractionated by Strong Cation Exchange (SCX) Fractionation. The lyophilized sample was resuspended in 1.5ml of 10mM ammonium formate, 25% acetonitrile, pH 3.0 and injected onto a 10 × 4.6-mm guard column attached to a polysulfoethyl A 200 × 4.6-mm (5-µm, 300-Å) column (Poly LC, Columbia, MD). The mobile phases consisted of solvent A (10mM ammonium formate, 25% acetonitrile, pH 3.0) and solvent B (500mM ammonium formate, 25% acetonitrile, pH 6.8). After sample loading, the separation was isocratic for 10 min with 100% solvent A with a flow rate of 1ml/min. Peptides were eluted using sequential linear gradients from 100% solvent A to 50% solvent B over 40 min and from 50% solvent B to 100% solvent B over another 10 min. The mobile phase was held at 100% solvent B for another 15 min. 1-ml fractions (1 min/fraction) were collected after the start of the gradient using a Shimadzu FRC-10A fraction collector (Kyoto, Japan) and combined into 30 fractions. Each fraction was lyophilized and analyzed by reversed-phase LC-FTICR. | |||
| PAe000330 | human serum fractionation/depletion methods, 01_glyco | 01_glyco | Human | Male, Serum, Sigma-Aldrich, catalog #H4522, lot #043K0502 | LTQ | Ammonium bicarbonate (50 mM) was added to achieve a final methanol concentration of 20% and the samples were digested with Trypsin Gold (Promega, Madison, WI) at a protein to enzyme ratio of 50:1 (w/w) at 37°C for 6 h. The samples were dried in a SpeedVac and resuspended in 50 mM ammonium bicarbonate prior to LC-MS analysis. | Human serum samples were denatured and reduced with 60% methanol and 10 mM dithiothreitol (DTT) at 60°C for 1 h and alkylated with 50 mM iodoacetamide (IAM) at room temperature in the dark for 30 min. | N-linked glycopeptide enrichment Briefly, 0.6 mL human serum was oxidized and coupled to 4 mL of Affi-Gel Hz Hydrazide Gel (50% slurry) (Bio-Rad, Hercules, CA). N-linked glycopeptides were released from the resin by adding 3µL PNGase F (glycerol free) (New England Biolabs, Inc., Beverly, MA) in 2 mL of 0.1 M freshly prepared NH₄HCO₃.The cleavage reaction was performed at 37°C with mixing overnight followed by washing with 2×2 mL of 80% ACN. The released glycopeptides were dried and resuspended in 0.4% acetic acid. | PMID:17269739 | ||
| PAe000336 | human serum fractionation/depletion methods, 22_mars | 22_mars | Human | Male, Serum, Sigma-Aldrich, catalog #H4522, lot #043K0502 | LTQ | Ammonium bicarbonate (50 mM) was added to achieve a final methanol concentration of 20% and the samples were digested with Trypsin Gold (Promega, Madison, WI) at a protein to enzyme ratio of 50:1 (w/w) at 37°C for 6 h. The samples were dried in a SpeedVac and resuspended in 50 mM ammonium bicarbonate prior to LC-MS analysis. | Human serum samples were denatured and reduced with 60% methanol and 10 mM dithiothreitol (DTT) at 60°C for 1 h and alkylated with 50 mM iodoacetamide (IAM) at room temperature in the dark for 30 min. | Multiple Affinity Removal System (MARS) column depletion The human Multiple Affinity Removal System (MARS) was purchased from Agilent Technologies (Palo Alto, CA) to deplete albumin, transferrin, IgG, IgA, anti-trypsin, and haptoglobin from human serum. Briefly, 100µL human serum was diluted 5-fold with Buffer A, filtered (0.22µm filter), and injected on the MARS column connected to a BioCad Vision HPLC system (Applied Biosystems, Foster City, CA). The flow-through was collected for 6 min (about 1.5 mL), desalted, and resuspended in 50 mM ammonium bicarbonate. | PMID:17269739 | ||
| PAe000115 | Human Erythroleukemia K562 cell line, PLC | Human Erythroleukemia K562 cell line huamn erythroleukemia K5632 cell line I think... | Human | K562 (erythroleukemia) | LTQ | K562 grown in suspension. | Proteins were alkylated with 14 mM iodoacetamide (Aldrich, Milwaukee, WI) for 30 min in the dark at room temperature. Reactions were quenched by adding 3 mM DTT, and proteins were immediately desalted on a PD10 column (Amersham, Piscataway, NJ) equilibrated with 100 mM NH₄HCO₃, followed by trypsinization at 37°C with 3% (w/w) trypsin (Wako, Richmond, VA, Catalog No. 20709891) added in 1% aliquots at t = 0, 4, and 12 h. The NH₄HCO₃ was removed by repeated lyophilization and resuspension in water (usually 3 times) until the conductivity after dilution of 10 µL of sample into 3 mL of water was less than 0.004 Ω⁻¹/cm. | Cells were washed twice by centrifugation, and pellets were flash frozen in liquid nitrogen. Cell pellets were suspended in lysis buffer (140mM potassium phosphate, pH 7.4, 150mM NaF, 1mM Na₃VO₄, 6mM EDTA, 6mM EGTA, 250mM NaCl, 4mM DTT) containing 40µg/mL leupeptin, 5µg/mL pepstatin A, 4mM benzamidine, and 20mM PMSF and sonicated 4 × 15 s at 4°C (Branson, microtip probe). Lysates were centrifuged at 200000g for 30 min at 4°C and soluble proteins recovered in the supernatant. Typically 10⁸ cells yielded ~15mg of protein. | Lyophilized peptides were dissolved in buffer A (5 mM K₂HPO₄, 5% acetonitrile pH 2.5) and fractionated by HPLC using a SCX column (PolySulfoethyl A, 2.1 mm i.d. × 200 mm, Poly LC), equilibrated in buffer A, and eluted using a gradient of increasing 0.5 M KCl in buffer A. | PMID:15228325 |
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