Proteins in cell lysates were digested with trypsin, labeled with Tandem Mass Tag reagents according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, MA, USA) and combined in equal amounts. An aliquot of the pooled sample was evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 μm, 2.1 mm × 150 mm; Waters, Elstree, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MS/MS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Data acquisition and processing was performed as described previously13 (link). For analysis, only rank 1 peptides and quantifications obtained using two or more unique peptides with high/medium confidence per protein were used. We selected a stringent comparative protein threshold of 2. Proteins that differed in level by twofold or more between replicate samples (due to heterogeneity between individuals) were excluded.
Tandem mass tag reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Tandem Mass Tag reagents are a set of isobaric labeling compounds designed for multiplexed quantitative proteomics. The reagents enable simultaneous identification and relative quantification of proteins across multiple samples in a single mass spectrometry experiment.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (3 mentions)
Orbitrap fusion tribrid mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Promega (2 mentions)
Xbridge beh c18 column, by Waters Corporation (1 mentions)
Ultimate 3000 liquid chromatography system, by Thermo Fisher Scientific (1 mentions)
Reversed phase c18 spe columns, by Waters Corporation (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
2695 hplc system, by Waters Corporation (1 mentions)
Pierce micro bca protein assay, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor tablet, by Roche (1 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Dylight 680 800 secondary antibodies, by Cell Signaling Technology (1 mentions)
Odyssey blocking buffer tbs, by LI COR (1 mentions)
Gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
Triethylammonium bicarbonate teab, by Merck Group (1 mentions)
Oasis hlb cartridge, by Waters Corporation (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Acetonitrile acn, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Fused silica capillary tubing, by Molex (1 mentions)
16 protocols using tandem mass tag reagent
1
Quantitative Mass Spectrometry-based Proteomics
2
Multiplexed Proteomics of Head and Neck Cancer
3
Mitochondrial Proteomic Sample Preparation
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Reduction, alkylation, trypsin digestion, and labeling of mitochondrial samples were performed according to the instruction of the manufacturer (Pierce™ Tandem Mass Tag Reagents, Thermo Scientific, USA). The peptides were labeled with TMT-labeled peptide samples and were pooled and subject to fractionation by using a PolyLC polysulfoethyl aspartamide column (100 mm × 2.1 mm, 5 μm, 300A pore size on Waters 2695 HPLC system) for off-line strong-cation exchange (SCX) chromatography fractionation. A gradient elution of 100% solvent A (10 mM monopotassium phosphate, 15% acetonitrile) to 100% solvent B (500 mM potassium chloride in solvent A) was performed in 40 min at 200 μl/min flow rate.
4
Quantitative Proteomic Analysis with TMT
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Samples were lysed in a buffer consisting of 50 mM Tris (pH 8.5), 8 M urea, 1% SDS, supplemented with protease and phosphatase inhibitors. Protein quantification was performed using Pierce™ Micro BCA Protein Assay (Thermo Scientific) according to the manufacture protocol. Disulfide bonds of the lysates were then reduced with and dithiothreitol (10mM) and the sulfhydryl groups were alkylated with iodoacetamide (10mM) and methanol/chloroform-precipitated and reconstituted in 100μl HEPES pH 8.5. Afterwards, proteins were digested with LysC (1:50; enzyme:protein) followed by digestion with trypsin (1:50; enzyme:protein) before quantifying, and 100 μg of the peptides of each sample was labeled with TMT reagent. Tandem mass tag reagents (Thermo Fisher Scientific) were dissolved according to manufacturer’s instructions. Samples were sent for TMT-based mass spectrometry and further processed at the Thermo Fisher Center for Multiplexed Proteomics (TCMP) facility at Harvard Medical School.
5
Subcellular Fractionation and Proteomic Analysis
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Cells were washed with cold PBS on ice, scraped off from six-well plates and centrifuged at 450g at 4°C for 3 min. The cells were then resuspended in 100 μl ice-cold buffer (10 mM Tris–Cl, pH 7.5, 150 mM NaCl, 3 mM MgCl2, and protease inhibitor) by vortexing. Next, 1.5 μl 10% NP-40 was added to the resuspension and incubated for 5 min on ice. The suspension was then centrifuged at 4,000g at 4°C for 5 min. The resulting supernatant represented the cytoplasmic fraction while nuclei were in the pellet. The pellet was resuspended in 100 μl buffer (10 mM Tris–Cl, pH 7.5, 150 mM NaCl, 3 mM MgCl2, and protease inhibitor) by tapping the tube. Nuclei were then centrifuged at 4,000g at 4°C for 5 min. Finally, the pellet was dissolved in lysis buffer for Western blot (to check the quality of nuclei fractionation from the cytoplasm) and mass spectrometry. Regarding mass spectrometry, the samples were labeled with Tandem Mass Tag reagents (Thermo Fisher Scientific) and analyzed with a Fusion Orbitrap Tribrid Mass Spectrometer (Thermo Fisher Scientific). Protein and peptide identification were performed with Integrated Proteomics Pipeline–IP2 (Integrated Proteomics Applications).
6
Antibody sourcing for protein detection
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Antibodies against RASA3 (sc-166442 and sc-34468), PLEKHA1 (sc-50468), ADAP1 (sc-47836) and GAPDH (sc-25778) were from Santa Cruz Biotechnology (Insight Scientific, Middlesex, UK). The antibody against PHLDB1 (HPA038448) was from Sigma Aldrich (Poole, UK). The antibody against DAPP1 (AF7024) was from R&D Systems (Abingdon, UK). The antibody against GST (2622) and DyLight 680/800 secondary antibodies were from Cell Signaling Technologies (New England Biolabs, Hitchin, UK). Anti-rabbit HRP-coupled secondary antibody was from Jackson ImmunoResearch (Stratech Scientific Ltd, Ely, UK). Immobilon FL polyvinylidene difluoride (PVDF) was from Merck Millipore (Dorset, UK). Odyssey blocking buffer (TBS) was from LI-COR Biosciences (Cambridge, UK). Blank control beads, PtdIns(3,4)P 2 beads (P-B034A), free PtdIns(3,4)P 2 (P-3408) and PIP Arrays (P-6100) were from Echelon Biosciences, distributed via Tebu-Bio (Peterborough, UK). cOmplete mini protease inhibitor tablets were from Roche Life Sciences (Welwyn Garden City, UK). Tandem Mass Tag reagents and NuPAGE sample buffer were from Thermo Fisher Scientific (Loughborough, UK). Leukocyte removal filters were from Pall (Portsmouth, UK). All other reagents were from Sigma Aldrich (Poole, UK) unless otherwise stated in the relevant methods subsection.
7
Multistep Brain Proteome Fractionation
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A three-part fractionation was used to decrease sample complexity prior to tryptic digestion and LC-MS analysis. As shown in Supplementary Figure S1 , frozen brain hemisphere was homogenized in modified phosphate buffered saline (NaCl concentration = 1M) with Halt Protease and Phosphatase Inhibitor Cocktail (PPIC) on ice in 10 s bursts. Following centrifugation at 20,000 × g at 4°C for 10 min the supernatant was mixed with chilled methanol (MeOH) for protein precipitation, while the pelleted material was saved and stored at −80°C as the “membrane” fraction for future use. The supernatants were incubated with chilled MeOH on ice for 30 min followed by centrifugation. All materials were then re-suspended in 20 mM triethylammonium bicarbonate (TEAB), pH 8.0 (Sigma Aldrich), 0.5% w/v sodium deoxycholate (SDC) via bath sonication. A process aliquot was taken to determine protein concentration using the bicinchoninic acid (BCA) assay (Pierce) and verified by SDS-PAGE and staining with Sypro Ruby. Samples were then prepared for tryptic digestion and labeling with Tandem Mass Tag (TMT) reagents (Thermo Fisher Scientific), 50 μg of each sample was then precipitated in four volumes of chilled acetone to concentrate and de-salt the material.
8
Urinary Proteome Profiling for EAE
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After collection, urine was immediately centrifuged at 2000g to remove pellets. Urinary proteins were extracted by adding three volumes of acetone. After centrifugation, proteins were dissolved in lysis buffer (8 M urea, 2 M thiourea, 25 mM dithiothreitol and 50 mM Tris). The urinary proteins were then denatured with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin (Promega) (1 : 50) at 37°C overnight using filter-aided sample preparation methods as previously described [12 (link)]. The digested peptides were desalted using Oasis HLB cartridges (Waters, USA).
Urine samples (from fifteen rats with EAE and fifteen control rats) collected on day 7 were used for MS analysis. As the tandem mass tag (TMT) reagents (Thermo Fisher Scientific) have six channels, the fifteen EAE samples were randomly divided into three TMT channels and the other controls samples were divided into the other TMT channels (total six group). Peptides in each sample were labelled with 126, 127, 128, 129, 130 and 131 according to the manufacturer's instructions. The labeled peptides were mixed and then analysed with two-dimensional LC-MS/MS.
Urine samples (from fifteen rats with EAE and fifteen control rats) collected on day 7 were used for MS analysis. As the tandem mass tag (TMT) reagents (Thermo Fisher Scientific) have six channels, the fifteen EAE samples were randomly divided into three TMT channels and the other controls samples were divided into the other TMT channels (total six group). Peptides in each sample were labelled with 126, 127, 128, 129, 130 and 131 according to the manufacturer's instructions. The labeled peptides were mixed and then analysed with two-dimensional LC-MS/MS.
9
Purification and Characterization of Recombinant Proteins
10
iTRAQ Proteomic Analysis of Barley
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An Isobaric tags for relative and absolute quantitation (iTRAQ) proteome experiment was conducted by Shanghai Bioprofile Technology Company. Briefly, peptides were labeled with Tandem Mass Tag (TMT) reagents according to the manufacturer’s instructions (Thermo Fisher Scientific, Logan, UT). Then, a TMT-labeled peptide mixture for nano-LC-MS/MS analysis was performed as previous described (54 (link)). The resulting liquid chromatography tandem mass spectrometry (LC-MS/MS) raw files were imported into MaxQuant software (version 1.6.0.16) for data interpretation and protein identification against the database UniProt_Hordeum-vulgare_201747-20180125 (downloaded on January 25, 2018, including 201,747 protein sequences), which was sourced from the protein database at https://www.uniprot.org/uniprot/?query=Hordeum+vulgare&sort=score .
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