TMT-10plex proteomics, HEK293 lysate, 5 conditions

{
    "experiment": {
        "id": 314,
        "custom_id": "MS-TMT-2026-04",
        "title": "TMT-10plex proteomics, HEK293 lysate, 5 conditions",
        "template": "Mass Spectrometry",
        "template_version": "v3.2",
        "category": "Analytical Chemistry",
        "status": "Complete",
        "started_on": "2026-06-08",
        "tags": [
            "mass-spectrometry",
            "proteomics",
            "TMT",
            "Orbitrap",
            "quantitative-proteomics"
        ]
    },
    "main_text": "This template captures a bottom-up TMT 10-plex quantitative proteomics study on cultured HEK293 cells across five conditions with two biological replicates per condition.\nGoal: identify and quantify differentially abundant proteins across five HEK293 treatment conditions, with TMT 10-plex labeling for multiplexed relative quantification and high-pH reverse-phase fractionation to maximize ID depth.\nMethod overview: Cell lysates (100 µg protein per sample) are reduced with DTT, alkylated with iodoacetamide, digested with trypsin overnight at 37°C (1:50 enzyme:substrate), desalted on C18 SPE cartridges, and labeled with TMT 10-plex reagents in 100 mM TEAB. Labeled peptides are pooled, fractionated into 12 high-pH reverse-phase fractions, and each fraction is analyzed on a Thermo Q Exactive HF-X via a 90-minute nano-LC gradient (300 nL/min) with HCD fragmentation at NCE 32%. Raw files are searched in MaxQuant 2.4 against UniProt human reviewed (SwissProt) with carbamidomethyl (C) as fixed, TMT (K, N-term) as label, oxidation (M) and acetyl (N-term) as variable, 1% FDR at PSM and protein level. Quantification uses reporter ion intensities corrected for isotope impurities. Downstream analysis runs in Perseus with median normalization, missing-value imputation, and limma-style differential abundance.\nExpected: 5,500-6,500 proteins identified at 1% FDR, 30,000-45,000 peptide IDs. 400-700 differentially abundant proteins at adjusted p < 0.05 across pairwise condition comparisons.",
    "extra_fields": {
        "Sample": {
            "Sample type": {
                "type": "select",
                "value": "cell lysate, tissue, plasma, secretome, IP eluate"
            },
            "Number of samples": {
                "type": "number",
                "value": "10"
            },
            "Protein input": {
                "type": "number",
                "value": "100 µg / sample"
            },
            "Digest enzyme": {
                "type": "select",
                "value": "trypsin, LysC, LysC+trypsin, GluC, AspN, chymotrypsin"
            }
        },
        "Labeling": {
            "Labeling chemistry": {
                "type": "select",
                "value": "TMT 10plex, TMT 16plex, TMT Pro, label-free, SILAC"
            },
            "TMT lot": {
                "type": "text",
                "value": "90111 · Lot WL323921"
            },
            "Pre-fractionation": {
                "type": "select",
                "value": "high-pH RP, SCX, none"
            },
            "Fractions collected": {
                "type": "number",
                "value": "12"
            }
        },
        "Acquisition": {
            "Instrument": {
                "type": "select",
                "value": "Q Exactive HF-X, Exploris 480, Astral, timsTOF Pro"
            },
            "LC column": {
                "type": "text",
                "value": "25 cm × 75 µm C18, 1.9 µm"
            },
            "LC gradient": {
                "type": "text",
                "value": "5-30% B over 90 min"
            },
            "Fragmentation": {
                "type": "select",
                "value": "HCD, EThcD, CID, ETD"
            }
        }
    },
    "attached_files": [
        {
            "filename": "TMT_labeling_scheme.pdf",
            "type": "pdf",
            "size": "184 KB"
        },
        {
            "filename": "instrument_method.meth",
            "type": "docx",
            "size": "36 KB"
        },
        {
            "filename": "proteinGroups.xlsx",
            "type": "xlsx",
            "size": "12.4 MB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Lyse cell pellets in 8M urea/100 mM TEAB + protease inhibitors",
            "duration": "0:30",
            "complete": true,
            "note": "10 samples · 100 µg each"
        },
        {
            "n": 2,
            "description": "BCA assay, normalize to 100 µg per sample",
            "duration": "0:45",
            "complete": true
        },
        {
            "n": 3,
            "description": "Reduce (DTT 5 mM, 60°C 30 min), alkylate (IAA 15 mM, 30 min RT dark)",
            "duration": "1:00",
            "complete": true
        },
        {
            "n": 4,
            "description": "Dilute urea to 1 M, trypsin digest 1:50 overnight at 37°C",
            "duration": "16:00",
            "complete": true
        },
        {
            "n": 5,
            "description": "Acidify with TFA, desalt on C18 SPE cartridges, lyophilize peptides",
            "duration": "2:00",
            "complete": true
        },
        {
            "n": 6,
            "description": "Resuspend peptides in 100 mM TEAB, label with TMT 10plex per channel",
            "duration": "1:30",
            "complete": true,
            "note": "See TMT scheme PDF"
        },
        {
            "n": 7,
            "description": "Quench labeling with hydroxylamine, pool all 10 channels equimolar",
            "duration": "0:30",
            "complete": true
        },
        {
            "n": 8,
            "description": "Offline high-pH RP fractionation on UltiMate 3000, collect 12 fractions",
            "duration": "2:00",
            "complete": true
        },
        {
            "n": 9,
            "description": "LC-MS/MS: each fraction on Q Exactive HF-X, 90-min nano-LC gradient",
            "duration": "24:00",
            "complete": true,
            "note": "12 fractions × 90 min + overhead"
        },
        {
            "n": 10,
            "description": "MaxQuant 2.4 search → Perseus → differential abundance analysis",
            "duration": "6:00",
            "complete": true,
            "note": "5,847 proteins · 516 DAPs FDR 5%"
        }
    ],
    "spreadsheet": [
        [
            "TMT Channel",
            "Sample",
            "Condition",
            "Bio rep",
            "Reporter intensity (median)",
            "Notes"
        ],
        [
            "126",
            "HEK293-A1",
            "control",
            "1",
            "4.82e7",
            "reference"
        ],
        [
            "127N",
            "HEK293-A2",
            "control",
            "2",
            "4.91e7",
            ""
        ],
        [
            "127C",
            "HEK293-B1",
            "siHER2",
            "1",
            "4.62e7",
            ""
        ],
        [
            "128N",
            "HEK293-B2",
            "siHER2",
            "2",
            "4.71e7",
            ""
        ],
        [
            "128C",
            "HEK293-C1",
            "lapatinib",
            "1",
            "4.55e7",
            ""
        ],
        [
            "129N",
            "HEK293-C2",
            "lapatinib",
            "2",
            "4.64e7",
            ""
        ],
        [
            "129C",
            "HEK293-D1",
            "IFN-γ",
            "1",
            "4.78e7",
            ""
        ],
        [
            "130N",
            "HEK293-D2",
            "IFN-γ",
            "2",
            "4.82e7",
            ""
        ],
        [
            "130C",
            "HEK293-E1",
            "combo",
            "1",
            "4.41e7",
            "mild ratio compression"
        ],
        [
            "131",
            "HEK293-E2",
            "combo",
            "2",
            "4.50e7",
            ""
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0410",
                "title": "HEK293 treatment series + lysate harvest",
                "owner": "A. Oduya",
                "date": "Apr 10"
            },
            {
                "ref_id": "EXP-2025-1108",
                "title": "MS facility instrument QC + suitability",
                "owner": "MS Facility",
                "date": "Nov 8 2025"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0614",
                "title": "TMT-10plex Bottom-Up Proteomics SOP v2.1",
                "owner": "QA Team",
                "date": "Jun 1"
            },
            {
                "ref_id": "DB-0102",
                "title": "UniProt human SwissProt + cRAP contaminants",
                "owner": "Refs DB",
                "date": "Mar 28"
            },
            {
                "ref_id": "METH-0207",
                "title": "Perseus differential abundance workflow v3",
                "owner": "Bioinf DB",
                "date": "Feb 10"
            }
        ]
    },
    "compounds": [
        {
            "name": "TMT 10plex Isobaric Label Reagent Set",
            "catalog_lot": "90111 · Lot WL323921",
            "stock_status": "In stock"
        },
        {
            "name": "Trypsin, Sequencing Grade Modified",
            "catalog_lot": "V5111 · Lot 0000489472",
            "stock_status": "In stock"
        },
        {
            "name": "Dithiothreitol (DTT)",
            "catalog_lot": "20290 · Lot 21F124501",
            "stock_status": "In stock"
        },
        {
            "name": "Iodoacetamide (IAA)",
            "catalog_lot": "I1149 · Lot SLBN3914",
            "stock_status": "In stock"
        },
        {
            "name": "C18 SPE cartridges (50 mg)",
            "catalog_lot": "WAT054925 · Lot 6342128",
            "stock_status": "In stock"
        },
        {
            "name": "EasySpray PepMap C18 column (25 cm)",
            "catalog_lot": "ES902 · Col SN 1284072",
            "stock_status": "Low"
        }
    ],
    "storage": [
        {
            "name": "Labeled peptide pool aliquots (10 µg)",
            "location": "-80 °C · Freezer C-1 · Drawer 1 · Box MS-2026-04",
            "count": "×12 fractions"
        },
        {
            "name": "Lysate stocks (original, 100 µL)",
            "location": "-80 °C · Freezer A-3 · Drawer 4 · Box LYS-04",
            "count": "×10"
        },
        {
            "name": "Raw .raw files (12 fractions)",
            "location": "NAS · /ms-data/2026-04/MS-TMT-2026-04/",
            "count": "×12, 6.2 GB total"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Proteomics Lab"
            },
            {
                "type": "Person",
                "name": "A. Oduya"
            },
            {
                "type": "Person",
                "name": "C. Iverson"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "A. Oduya"
            },
            {
                "type": "Person",
                "name": "C. Iverson"
            },
            {
                "type": "Person",
                "name": "Dr. T. Williams"
            }
        ]
    }
}