Ciliary axonemes were isolated from the surface of highly ciliated airway cells by application of cilia buffer as described, with some modifications (64 (link)) as described in the Supplemental Methods . TMT labeling was performed using the TMT 10-plex reagent kit (Thermo Fisher Scientific). Detailed methods of liquid chromatography–mass spectroscopy analysis is included in the Supplemental Methods .
Tmt 10 plex reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TMT 10-plex reagent kit is a set of isobaric labeling reagents used for quantitative proteomics analysis. The kit contains 10 different reporter ions that can be used to label peptides from up to 10 different samples, allowing for simultaneous identification and quantification of proteins across multiple conditions.
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4 protocols using tmt 10 plex reagent kit
1
Proteomic Analysis of Ciliary Axonemes
2
Quantitative Phosphoproteomics of Gastric Cancer
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For quantitative phosphoproteomics experiment, a total of 9 samples were used-3 gastric cancer tissue samples, 3 case-matched adjacent normal gastric tissue samples, and 3 case-matched xenograft samples. The digested samples were labeled using TMT 10-plex reagent kit (Thermo Scientific, Bremen, Germany) as described previously [18] . Digested proteins derived from the three adjacent normal gastric tissue were labeled with TMT labels, 127N, 128C and 130N, and the case-matched gastric tumor were labeled with 127C, 129N, and 130C and PDX protein samples were labeled with 128N, 129C and 131, respectively. The labeled samples were pooled and loaded on a Sep-Pak C18 column (Waters, Milford, MA) equilibrated with 0.1% trifluoroacetic acid (TFA). Washed peptides were eluted using 0.1% TFA and 40% acetonitrile (ACN) and subject to lyophilisation. The lyophilized samples were reconstituted in 7 mM TEABC (pH 9, solution A) and basic pH reverse phase chromatography (bRPLC) was carried out as described previously [19] . The bRPLC fractions were pooled to obtain 12 fractions. Further sample preparation details, including phosphopeptide enrichment, LC-MS/MS parameters, and data analysis are provided in Supplementary Methods.
3
Pulmonary Artery Protein Extraction and Labeling
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Pulmonary artery samples were ground into powder in liquid nitrogen and extracted with lysis buffer. Two milliliters of lysis buffer (7 M urea, 4% SDS, 1× Protease Inhibitor Cocktail [Roche Ltd. Basel, Switzerland]) was added to each sample, followed by sonication on ice and centrifugation at 13,000 rpm for 10 min at 4 °C. The protein concentration of the supernatant was determined using the BCA protein assay, 100 µg of protein per condition was transferred into new tubes, and the final volume was adjusted to 100 µl with 100 mM TEAB. To each sample, 5 µl of 200 mM DTT was added, and samples were incubated at 50 °C for 1 h. Then, 5 µl of 375 mM iodoacetamide was added, and the samples were incubated for 30 min at room temperature, protected from light. For each sample, proteins were precipitated with ice-cold acetone and were then redissolved in 100 µl of 100 mM TEAB. Then, the proteins were digested with sequence-grade modified trypsin (Promega, Madison, WI), and the resultant peptide mixture was labeled using chemicals from the 10-plex TMT Reagent Kit (Thermo Scientific, USA). The labeled samples were combined and desalted using a C18 SPE column (Sep-Pak C18, Waters, Milford, MA).
4
Tryptic Peptide Labeling and Quantification
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Protein digestion was performed according to the filter-aided sample preparation (FASP) method (Wiśniewski et al., 2009 (link)). Briefly, the proteins were digested with trypsin (enzymes/substrate ratio 1:50) overnight after reductively alkylation with 10 mM dithiothreitol (DTT) (1 h, 60°C) and 50 mM iodoacetamide (IAA) (40 min, room temperature). The digested peptides were lyophilized and resolved in 200 mM triethylamine buffer (TEAB). Finally, the lyophilized samples were labeled using the 10 plex TMT reagent kit (Thermo scientific, USA) following the manufacturer's instructions. Briefly, the tryptic peptides (100 μg each) were labeled with TMT 10-plex with 126-tag (21d-1), 127N-tag (21d-2), 127C-tag (21d-3), 128N-tag (27d-1), 128C-tag (27d-2), 129N-tag (27d-3), 129C-tag (40d-1), 130N-tag (40d-2), 130C-tag (40d-3), and 131-tag (MIX). Proteomics platform was provided by Shanghai Luming Biotech. Co., Ltd. (Shanghai, China).
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