| Metadata |
| datasetIdentifier | PASS00470 |
| datasetType | MSMS |
| submitter | Michael Shortreed <mshort@chem.wisc.edu> |
| submitter_organization | UW Madison-WI, Department of Chemistry |
| lab_head_full_name | Lloyd Smith |
| lab_head_email | smith@chem.wisc.edu |
| lab_head_organization | UW Madison-WI, Department of Chemistry |
| lab_head_country | United States |
| datasetTag | B6_CAST_ISLET001 |
| datasetTitle | 9 each Velos Orbitrap injections of high pH fractionated B6 and CAST mouse islet lysate |
| publicReleaseDate | 2015-04-09 00:00:00 |
| finalizedDate | 2015-04-09 09:32:10 |
| summary | Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify all posttranslational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables Global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified >1000 unique, high-confidence modified peptides comprising 31 different PTM types in single-pass database searches |
| contributors | Michael Shortreed, Mark Keller, Lloyd Smith |
| publication | Shortreed, MR; Wenger, C; Frey, BL; Sheynkman, G.; Smith, LM; Global Identification of Protein PTMs in a Single-pass Database Search, Nature Methods, submitted |
| growth | Male C57BL/6J (B6) and CAST/EiJ (CAST) mice were purchased from The Jackson Laboratories (Bar Harbor, Maine) and housed in an environmentally controlled vivarium at the University of Wisconsin Biochemistry Department. Mice were provided standard rodent chow (Purina no. 5008) and water ad libitum, and maintained on a 12-hour light/dark cycle (6 AM – 6 PM). At 10 weeks of age, mice were sacrificed by CO2 asphyxiation. All animal procedures were preapproved by the University of Wisconsin Animal Care and Use Committee. |
| treatment | n/a |
| extraction | Intact pancreatic islets were isolated using a collagenase digestion procedure as previously described.[ Am J Physiol Endocrinol Metab, 2005. 289(2): p. E218-24] Briefly, 5 ml of collagenase type XI (Sigma), dissolved in Hanks’ balanced salt solution (Gibco), was injected into the pancreas via the common bile duct (0.45 mg/ml with 0.02% BSA). The pancreas was removed and incubated at 37°C for 16 min with intermittent agitation. A ficol gradient was used to partially purify islets from the digested pancreas, followed by further purification by manual picking under a stereomicroscope. During the purification procedure, islets were maintained in Krebs-Ringer bicarbonate buffer containing (mM): 118.41 NaCl, 4.69 KCl, 2.52 CaCl2, 1.18 MgSO4, 1.18 KH2PO4, 25 NaHCO3 and 5 HEPES supplemented with 0.2% BSA and 16.7 mM glucose. Islets from two B6 mice (and separately two CAST mice) were pooled and then apportioned for RNA-Seq (250 islets per sample) and protein analyses (remainder: 400 B6 islets, 470 CAST islets). For RNA profiling, whole islet RNA was purified as previously described yielding ~1 µg total RNA.[21] For protein analyses, islets were washed 3 times in ice-cold PBS, gently pelleted and snap frozen in liquid nitrogen.
Protein was extracted and then digested into peptides using an adaptation of the filter aided sample preparation (FASP) procedure.[Mol Cell Proteomics, 2013. 12(8): p. 2341-53; Nat Methods, 2009. 6(5): p. 359-62] Islets were thawed on ice, and the protein extracted with 90 µL of SDT lysis buffer, consisting of 4% sodium dodecyl sulfate (SDS, Bio-Rad), 0.1 M Tris-HCl (pH 7.6, Teknova), and 0.1 M dithiothreitol (DTT, Sigma). The mixtures were incubated at 95 °C for 6 min with intermittent vortexing. The resulting lysates were cooled in an ice bath, followed by bath sonication (Fisher Scientific FS20) for three cycles of 20 s with ice bath cooling during 20 s rest periods. Debris was pelleted by centrifugation at 16,000 g for 5 min., and 85 µL of supernatant was collected.
Removal of detergents and salts was accomplished by FASP, using multiple washes in a 30K MWCO filter (Vivacon 500 from Sartorius). The 85 µL protein extract was diluted with 570 µL of 8 M urea (Sigma), 0.1 M Tris-HCl pH 8.0. Half of this solution was centrifuged at 14,000 g through the filter (25 min.), followed by the other half (25 min.), and then 0.2 mL urea/Tris wash (20 min.). The proteins were alkylated in the filter with 0.1 mL of 0.05 M iodoacetamide (Sigma) in urea/Tris for 20 min. at room temperature in the dark, followed by centrifugation (15 min.). The proteins were washed three times with 0.1 mL of urea/Tris and three times with 0.1 mL of 0.05 M ammonium bicarbonate (ABC, Fluka) with 15 min. centrifugations for each wash. Tryptic digestion was performed at 37 °C overnight on the proteins in the filter by addition of 2 µg trypsin (Promega) in 75 µL of 0.05 M ABC. Peptides were collected by centrifugation (10 min.) into new collection tubes, followed by washing the remaining peptides out of the filter with 40 µL ABC, 10 min. centrifugation, 50 µL 0.5 M NaCl, and a final 10 min. centrifugation. |
| separation | The tryptic peptide digests were fractionated using high pH reverse-phase chromatography on a Shimadzu HPLC system (LC-10AD, SCL-10A VP, SPD-10A VP, Shimadzu, Columbia, MD) and a Phenomenex C18 Gemini 3µ, 110Å, 3.0 x 150 mm column (Phenomenex, Torrance, CA). The high pH method was adopted from Gilar et al.[ Nat Methods, 2009. 6(5): p. 359-62] Mobile phase A (MPA) was 20 mM ammonium formate, pH 10, and B (MPB) was 20 mM ammonium formate, pH 10, in 70% acetonitrile. The HPLC flow was 1.0 mL/min and the gradient was: 0% MPB isocratic for 15 min (trapping step), linear ramp to 100% MPB over 60 min, hold at 100% MPB for 5 min, to 0% MPB over 2 min, and equilibration at 0% MPB for 20 min. Fractions were collected every minute using a Gilson 203 fraction collector (Gilson, Middleton, WI) for a total of 9 fractions collected during the range of peptide elution as discernable from the UV-Vis trace. By comparison of the UV-Vis trace to that of a standard, it was estimated that the B6 and CAST samples contained 34 and 33 µg of peptides, respectively. Fractions were dried down using vacuum centrifugal concentration (Savant Speed- Vac, Thermo, Pittsburgh, PA) and stored at -80°C. |
| digestion | Please see the Extraction Protocol for the description of tryptic digestion with FASP. |
| acquisition | Each fraction was reconstituted in 10 µL of 5% acetonitrile and 1% formic acid in water, and then between 4 and 9.5 µL of each fraction was analyzed by LC-MS (using the above UV-Vis trace to estimate peptide content of each fraction and avoid injecting more than 2.5 µg of peptides per run). The HPLC-ESI-MS/MS system consisted of a Waters nanoAcquity HPLC (Milford, MA) connected to an electrospray ionization (ESI) ion-trap/orbitrap mass spectrometer (LTQ Orbitrap Velos, Thermo Scientific, San Jose, CA). The LC column was prepared by packing 20 cm of 3µm MAGIC aqC18 beads (Bruker-Michrom, Auburn, CA) into a 100µm i.d. capillary whose tip was pulled to ~1 µm with a P-2000 laser puller (Sutter Instruments, Novato, CA). The full HPLC method was 195 min long at a flow rate of 0.3 µL/min, and it included a 124 min gradient from 2% to 30%, with a brief ramp to 70%, mobile phase B (0.1% formic acid in acetonitrile) with the remainder being mobile phase A (0.1% formic acid in water). A full-mass scan (300-1500 m/z) was performed in the orbitrap at a resolution of 60,000. The ten most intense peaks with z > 1 from the full scan were selected for fragmentation by higher-energy collisional dissociation (HCD, collision energy = 42). The isolation width for the precursor ions was 3.0 m/z, and the product ions from fragmentation were analyzed in the orbitrap detector at a resolution of 7500. Dynamic exclusion was enabled with a repeat count of two over 30 s and an exclusion duration of 120 s. XCalibur software version 2.1.0 was used for data collection. |
| informatics | Protein FASTA and XML files were obtained from the UniProt repository. The database used for the Jurkat samples was limited to those proteins with mRNA transcript abundances exceeding 0.1 Transcripts Per Million[Mol Cell Proteomics, 2013. 12(8): p. 2341-53]. The database used for the mouse samples used the UniProt Mus musculus reference protein set supplemented with the list of common contaminants available at the following URL ftp://ftp.thegpm.org/fasta/cRAP.
The software program Morpheus was used for all database searching. It can be obtained at http://morpheus-ms.sourceforge.net/. For this work, it was modified to accept UniProt XML in addition to FASTA protein databases. When a UniProt XML database is specified, all curated modifications are extracted. Details of each modification (name, mass shift, target) are read from a local copy of http://www.uniprot.org/docs/ptmlist. All valid modifications are added to the variable modifications box in the Morpheus graphical user interface with the prefix “UniProt: “ and selected by default. During each search, all protein sequences are read along with the locations of selected UniProt variable modifications. UniProt modifications are reversed along with the protein sequence during generation of decoys.
A critical routine in the code takes a base peptide sequence and generates all the isoform combinations possible given the variable modifications selected, up to a user-defined limit (1024 by default). This code was modified slightly to consider UniProt modifications only at their previously observed location. Otherwise the logic for combinatorially generating all possible peptide isoforms is identical.
The following settings were used in all searches: Protease = trypsin (no proline rule); Maximum Missed Cleavages =2; Initiator Methionine Behavior =variable; Fixed Modifications = carbamidomethylation of C; Variable Modifications = oxidation of M; Maximum Variable Modification Isoforms Per Peptide = 1024; Precursor Mass Tolerance = ±2.1 Da (monoisotopic); Precursor Monoisotopic Peak Correction = disabled; Product Mass Tolerance = ±0.01 Da (monoisotopic); Maximum False Discovery Rate = 1%. FASTA_vP searches used additionally variable phorphorylation of S, T and Y. G-PTM searches used XML database files rather than FASTA files. Counts of posttranslational modified peptides do not include the oxidation of methionine or the carbamidiomethylation of cysteine as these occur during sample handling and therefore are somewhat uninteresting. The list of identified proteins including numbers of PSMs (total and modified) are provided in Supplementary Table 6 for both Jurkat and mouse, where B6 and CAST mouse data are segregated to allow for their comparison. |
| instruments | Thermo Scientific Velos Orbitrap |
| species | Mouse Male C57BL/6J (B6) |
| massModifications | static: C+57.021464, variable: M+15.9949, several others as variable (see above) |
