Sample processing and LC–MS/MS analysis were performed as reported previously [10 (link)]. Briefly, proteins extracted from fresh frozen pancreas specimens were reduced, alkylated and digested into peptides using Lys-C and trypsin. The peptides were analyzed using a high-performance liquid chromatography system, EASY-nLC 1000 connected to Q Exactive quadrupole-Orbitrap mass spectrometer equipped with a nanospray ion source (Thermo-Fisher Scientific, Bremen, Germany). To identify the detected proteins, the acquired MS/MS data were managed using Proteome Discoverer software, version 1.4 (Thermo Fisher).
Proteome discoverer software version 1
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
Proteome Discoverer software version 1.4 is a data processing and analysis software tool designed for mass spectrometry-based proteomics research. The software's core function is to facilitate the identification and quantitation of proteins from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Q exactive quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Easy spray source, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000 hplc system, by Thermo Fisher Scientific (1 mentions)
Precleared protein a g beads, by Roche (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (1 mentions)
Picotip emitter, by New Objective (1 mentions)
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Velos pro, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Multiscreen 96 well filter plate, by Merck Group (1 mentions)
Ltq orbitrap xl etd hybrid ion trap orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Sypro ruby protein gel stain, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
Itraq 8 plex kit, by AB Sciex (1 mentions)
10 protocols using proteome discoverer software version 1
1
Pancreas Protein Extraction and LC-MS/MS
2
Proteome Discoverer SEQUEST HT Database Search
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Thermo Scientific Proteome Discoverer software version 1.4 was used to search MS/MS spectra against the Swiss-Prot human database (http://www.uniprot.org/downloads ) using SEQUEST HT search engine. Static modifications included carbamidomethylation (C) and Dynamic modifications included methionine oxidation and deamidation of Asparagine and Glutamine. The mass tolerances of the precursor ion and the fragment ions were set to 10 ppm and 0.6 Da, respectively. Full tryptic specificity was required with up to two miss cleavage Sites. Resulting peptide hits were filtered for maximum 1% FDR using the Percolator algorithm and with the minimum number of two peptides per protein.
3
Proteomic Analysis of Extracellular Vesicles
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EVs (10 μl with ≈ 60 μg total protein) were electrophoresed into the resolving gel of a 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) for 1 cm. The samples were then stained with colloidal Coomassie Blue G-250, cut from the gel, and subjected to mass spectrometry analysis and protein identification by an external service (CEQUIBIEM proteomic service, Buenos Aires, Argentina). All proteomic analyses were performed as described previously [16 ]. Samples were processed using nano-HPLC [high-performance liquid chromatography] (EASY-Spray Accucore, Thermo Scientific, West Palm Beach, FL, USA) coupled to a mass spectrometer with Orbitrap technology (Q Exactive, Thermo Scientific, West Palm Beach, FL, USA). The resulting data were analyzed using Proteome Discoverer software version 1.4 (Thermo Scientific). Further analyses were performed using proteins with at least two peptides in duplicate.
4
Quantitative Mouse Proteome Analysis
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A EASY-nLC 1000 HPLC system and EASYSpray
source (Thermo Scientific) with EASY-Spray PepMap RSLC C18 25 cm ×
75 μm ID column (Thermo Scientific) were used to separate peptides
with a 5–25% (v/v) ACN gradient in 0.1% (v/v) formic acid over
120 min at a flow rate of 300 nL/min. Samples were analyzed on Orbitrap
Elite and LTQ Orbitrap XL mass spectrometers (Thermo Scientific) using
top 15 Fourier transform (FT) MS/MS with higher-energy collision dissociation
(HCD) or top 3/3 ion-trap collision-induced dissociation (CID)/FT
HCD experiments.
Proteome Discoverer software version 1.4 (Thermo
Scientific) was used to search MS/MS spectra against the Swiss-Prot
mouse database using the SEQUEST HT or Mascot 2.3 search engines.
Dynamic modifications included carbamidomethylation (C), iodoTMTsixplex
(C), and methionine oxidation. Resulting peptide hits were filtered
for a maximum 5% false discovery rate using the Percolator. The iodoTMTsixplex
quantification method within Proteome Discoverer software was used
to calculate the reporter ratios with a mass tolerance ±10 ppm.
source (Thermo Scientific) with EASY-Spray PepMap RSLC C18 25 cm ×
75 μm ID column (Thermo Scientific) were used to separate peptides
with a 5–25% (v/v) ACN gradient in 0.1% (v/v) formic acid over
120 min at a flow rate of 300 nL/min. Samples were analyzed on Orbitrap
Elite and LTQ Orbitrap XL mass spectrometers (Thermo Scientific) using
top 15 Fourier transform (FT) MS/MS with higher-energy collision dissociation
(HCD) or top 3/3 ion-trap collision-induced dissociation (CID)/FT
HCD experiments.
Proteome Discoverer software version 1.4 (Thermo
Scientific) was used to search MS/MS spectra against the Swiss-Prot
mouse database using the SEQUEST HT or Mascot 2.3 search engines.
Dynamic modifications included carbamidomethylation (C), iodoTMTsixplex
(C), and methionine oxidation. Resulting peptide hits were filtered
for a maximum 5% false discovery rate using the Percolator. The iodoTMTsixplex
quantification method within Proteome Discoverer software was used
to calculate the reporter ratios with a mass tolerance ±10 ppm.
5
Immunoprecipitation and Proteomics Analysis
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DLD-1 cell lysates were obtained, and the total protein concentration were measured. In brief, 2 mg of protein lysate, 30 μl of precleared protein A/G beads (Roche, Switzerland) and 1 μg of primary antibody (rabbit IgG or TRIM29) were incubated together at 4° C overnight. Proteins were then separated by SDS–PAGE, and silver staining was performed after gel electrophoresis. The experiments were repeated three times, and a significant band at 58 KDa (which was identified as PKM1/2) was observed compared to the corresponding band in the control group. The following LC-MS/MS procedures to identify proteins from the samples were carried out by Capitalbio Technology (Beijing, China).
Proteome discoverer software (version 1.4) (Thermo Scientific, USA) was used to perform database searches against the Oryctolagus cuniculus database (46601 proteins) using the Sequest algorithms. The following criteria were applied: precursor mass tolerance of 15 ppm and a fragment mass tolerance of 20 mmu. The results were filtered using the following settings: high confident peptides with a global FDR<1% based on a target-decoy approach were included in the results (Table 1 ).
Proteome discoverer software (version 1.4) (Thermo Scientific, USA) was used to perform database searches against the Oryctolagus cuniculus database (46601 proteins) using the Sequest algorithms. The following criteria were applied: precursor mass tolerance of 15 ppm and a fragment mass tolerance of 20 mmu. The results were filtered using the following settings: high confident peptides with a global FDR<1% based on a target-decoy approach were included in the results (
6
Proteomic and Transcriptomic Profiling of Patient Samples
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Proteomic and transcriptomic data were generated from 71 samples from the above-mentioned patients. The pathological sections were reviewed by three pathologists to validate the diagnosis before sequencing. All specimens were stored at − 80 °C until protein and RNA isolation, and sequencing was performed by Beijing CapitalBio Technology Inc.
The experimental steps are described in theSupplementary Methods . Briefly, specimens were lysed using protein extraction buffer (8 M urea,0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Biotechnology, China) and protease inhibitor cocktail (Roche, USA). Tandem mass tags TMTpro (Pierce, USA) with different reporter ions (126–131 Da) were applied as isobaric tags for relative quantification and TMT labeling was performed following the manufacturer’s instructions. The MS analysis was conducted using an Q Exactive mass spectrometer (Thermo Scientific, USA). Proteome discoverer software (version 1.4) (Thermo Scientific, USA) was used to perform database searching against the RefSeq database. The results were filtered using the following settings: high confident peptides with a global FDR < 1% based on a target-decoy approach. The proteomic data have been uploaded into the iProX database (https://www.iprox.org ); (project ID IPX0004253000).
The experimental steps are described in the
7
LC-ESI-MS/MS Workflow for Protein Identification
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LC-ESI-MS/MS was conducted using a nanoflow HPLC system (UltiMate 3000, Dionex) connected to a hybrid ion trap-orbitrap highresolution tandem mass spectrometer (Velos Pro, Thermo Scientific) operated in data-dependent acquisition (DDA) mode. Each sample (1 μL) was injected onto a capillary column (C18 Onyx Monolithic, 0.10 mm × 150 mm, Phenomenex) at a flow rate of 300 nL/min. Samples were sprayed at 1.6 kV using fused silica noncoated emitters (20 μm inside diameter with a 10 μm ID tip, PicoTip Emitter from New Objective). Chromatographic separation was achieved using a linear gradient from 3 to 35% solvent B in solvent A over 30 min and then increasing the level of solvent B to 95% over 5 min (solvent A consisted of 0.1% formic acid in water and solvent B 0.1% formic acid in acetonitrile). The raw data files were acquired (Xcalibur, Thermo Fisher) and exported to Proteome Discoverer software version 1.4 (Thermo Fisher) for peptide and protein identification using the Sequest database search algorithm. 27 Carbamidomethyl cysteine and oxidized methionines were selected as static and dynamic modifications, respectively. Peptide to spectrum match (PSM) validation was conducted using high-confidence XCorr threshold values of 1.9, 2.3, and 2.6 for doubly, triply, and more highly charged precursors.
8
Proteomic Profiling of Purified Enzymes
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzymes was performed with SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific). The protein bands were excised and digested with trypsin (Promega, Madison, WI) in a MultiScreen 96-well filter plate (Merck Millipore, Darmstadt, Germany). The tryptic digests were analyzed with nano-LC (Shimadzu, Kyoto, Japan) and an LTQ OrbitrapXL ETD Hybrid Ion Trap-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) and identified using the Mascot database search system (Matrix Science, Boston, MA) and the SProt database. Data processing was performed by Proteome discoverer software version 1.4 (Thermo Fisher Scientific). The precursor mass tolerance was set at 20 ppm, the fragment ion mass tolerance was 0.2 Da, and the false discovery rate was 0.01.
9
Quantitative Proteomics Analysis by iTRAQ
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First, iTRAQ tagging and analysis was performed. The protein from each sample was reduced, alkylated and digested with trypsin. The digested peptides were dried and reconstituted in 50 µl 1 M triethylammonium bicarbonate. The dried peptides were labeled following the manufacturer's protocols (iTRAQ 8-plex kits; AB Sciex LLC, Framingham, MA, USA). Second, all the labelled peptides were pooled together followed by the separation of fractions using reversed-phase liquid chromatography which was performed by a RIGOL L-3000 system (RIGOL Technologies, Inc., Beijing, China). Then the fractionated peptides were analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) fitted with a nano-liquid chromatography system. Then Proteome Discoverer software version 1.3 (Thermo Fisher Scientific, Inc.) was used to interpret the data files. The files were searched using the Mascot search engine against the human protein database downloaded from NCBI (www.ncbi.nlm.nih.gov , Refseq. Human.20130704. fasta).
10
Identification of TANGO10 Interactors
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Transfected S2 cells and transgenic fly heads (tim-GAL4/UAS-Tango10-3xFLAG, elav-GAL4/Y; UAS-Tango10-3xFLAG/+) expressing triple FLAG-tagged TANGO10 were harvested and immunoprecipitated with anti-FLAG beads at ZT10 or ZT22, as in ref. 6 (link), with n = 1 experiment per timepoint for each GAL4. Bound proteins were eluted using 3xFLAG peptides (Sigma). The eluted samples were subject to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis by the Northwestern Proteomics Core Facility. The proteomics hits were identified from analysis using Proteome Discoverer software version 1.3 (Thermo Scientific), with hits for each GAL4 defined as proteins identified from either or both timepoints. Proteins identified in any GAL4-only controls were excluded. Proteins identified in proteomic analysis of FLAG-tagged TWENTY-FOUR in fly heads (6 (link)) were also removed from the hit list to improve specificity.
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