Dataset Information

Large-scale Proteomics Profiling of Peripheral Blood of DM1 patients identifies biomarkers for disease severity and functional capacity

ABSTRACT: 3 µl of each human serum sample was added to 40 µl lysis buffer containing 5% sodium dodecyl sulfate (SDS) and 50 mM triethylammonium bicarbonate (TEAB), pH 8.5. Proteins were reduced by addition of 15 mM dithiothreitol and incubation for 30 minutes at 55˚C and then alkylated by addition of 30 mM iodoacetamide and incubation for 15 minutes at RT in the dark. Phosphoric acid was added to a final concentration of 1.2% and subsequently samples were diluted 7-fold with binding buffer containing 90% methanol in 100 mM TEAB, pH 7.55. The samples were loaded on the 96-well S-Trap™ plate (Protifi), placed on top of a deepwell plate, and centrifuged for 2 min at 1,500 x g at RT. After protein binding, the S-trap™ plate was washed three times by adding 200 µl binding buffer and centrifugation for 2 min at 1,500 x g at room temperature. A new deepwell receiver plate was placed below the 96-well S-Trap™ plate and 50 mM TEAB containing trypsin (1/100, w/w) was added for digestion overnight at 37°C. Using centrifugation for 2 min at 1,500 x g, peptides were eluted in three times, first with 80 µl 50 mM TEAB, then with 80 µl 0.2% formic acid (FA) in water and finally with 80 µl 0.2% FA in water/acetonitrile (ACN) (50/50, v/v). Eluted peptides were dried completely by vacuum centrifugation. Dried peptides were redissolved in 100 µl 0.1% FA, the peptide concentration was determined on a Lunatic spectrophotometer (Unchained Labs) and was adjusted to 0.015 µg/µl with 0.1% FA1. iRT peptides (Biognosys, P/N Ki-3002-1) were added to all samples according to the manufacturer’s instructions. Then, 300 ng of each sample was loaded on Evotips (Evosep, P/N EV2003) according to the manufacturer’s instructions with a substitution of the wash step after sample loading by two wash steps with 80 µl 0.1% FA. All loaded Evotips were stored in 0.1% FA at 4°C until LC-MS/MS analysis was started.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

DISEASE(S): Myotonic Dystrophy Type 1

SUBMITTER: Tine Claeys

LAB HEAD: Peter A.C. ’t Hoen

PROVIDER: PXD067476 | Pride | 2026-06-26

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	H088664_1p_Evo15_1p9_150umID_PRCRT-111_plasma_300ng_DIA_iRT_2A1.raw	Raw
	H088665_1p_Evo15_1p9_150umID_PRCRT-111_plasma_300ng_DIA_iRT_2A2.raw	Raw
	H088666_1p_Evo15_1p9_150umID_PRCRT-111_plasma_300ng_DIA_iRT_2A3.raw	Raw
	H088667_1p_Evo15_1p9_150umID_PRCRT-111_plasma_300ng_DIA_iRT_2A4.raw	Raw
	H088668_1p_Evo15_1p9_150umID_PRCRT-111_plasma_300ng_DIA_iRT_2A5.raw	Raw

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Project description:Sample preparation was conducted using an adaptation of our SimPLIT workflow10. Four cell pellets corresponding to four different colorectal cancer cell lines were lysed in a buffer containing 1% sodium deoxycholate (SDC, Merck, Cat. No. 30970), 100 mM triethylammonium bicarbonate (TEAB, Merck, Cat. No. T7408), 10% isopropanol and 50 mM NaCl, freshly supplemented with 5 mM TCEP (Thermo, Bond-breaker Cat. No 77720), 10 mM iodoacetamide (IAA, Merck, Cat. No. I1149), universal nuclease 1:2000 vol/vol (Pierce, Cat. No. 88700) and Halt protease and phosphatase inhibitor cocktail (Thermo, Cat. No. 78442, 100X) with 5 min of bath sonication. Protein concentration was measured with the Quick Start Bradford protein assay (Bio-Rad, Cat. No. 5000205). Aliquots of 30 μg of total protein from each cell line were transferred in a 96-well plate according to the 58-plex design. An E. coli protein extract (Bio-Rad, Cat. No. 1632110) was added at 1 μg or 0.5 μg across the human cell lysates. The plate was dried and the samples for the arginine-specific cleavage set (TrypR) were reconstituted in 25 μL (TMT11 subset) or 12.5 μL (TMT18 subset) of 100 mM TEAB buffer followed by the addition of 10 μL TMT11 (stock 20 μg/μL, Thermo Fischer Scientific, Cat. No. A34808) or 5 μL TMTpro18 (stock 25 μg/μL, Thermo Fischer Scientific, Cat. No. A52045), and 1h reaction at room temperature for protein-level TMT labelling. The TMT labelled samples were SpeedVac dried, and all samples (labelled and unlabelled) were reconstituted in 20 μL of 100 mM TEAB buffer. Proteins were digested overnight at room temperature by the addition of 2 μL LysC (Thermo Fischer Scientific, Cat. No. 90307) or Trypsin (Thermo Fischer Scientific, Cat. No. 90059) stock solution of 500 ng/μL in 0.1% formic acid (final enzyme 50 ng/μL, 1:30) for the LysC and TrypR sets respectively. The peptides were then fully labelled by another addition of 10 μL TMT11 or 5 μL TMTpro18. The reaction was quenched after 1h with the addition of 2 μL 5% hydroxylamine solution and premix samples were prepared by pooling 0.5 μL from each sample into 100 μL of 0.1% TFA for each sub-plex separately. In each premix, SDC was precipitated with addition of formic acid at 2% and centrifugation at 20,000 × g for 5 minutes. Supernatants were transferred into clean vials for LC-MS analysis. Following evaluation of the premix runs, samples were pooled into four sub-plexes, diluted with 100 μL of HPLC water, heated at 95 °C for 5 min, cooled down and heated for another 5 minutes. Lastly, each pool was acidified with formic acid at 2%, the precipitated SDC was removed by centrifugation and supernatants were SpeedVac dried.High-pH Reversed-phase Peptide FractionationAll four sub-plexes were pooled in 100 μL of 0.1% (v/v) ammonium hydroxide and the final peptide pool was fractionated with high pH Reversed-Phase chromatography using the XBridge C18 column (2.1 × 150 mm, 3.5 μm, Waters) on an UltiMate 3000 HPLC system over a 1% gradient in 35 min. Mobile phase A was 0.1% (v/v) ammonium hydroxide and mobile phase B was 0.1% ammonium hydroxide (v/v) in acetonitrile. Fractions were collected every 30 sec and eventually pooled into 45 samples for LC-MS analysis.

Project description:Proteomic analysis of chick vestibualr hair bundles purified using the twist-off method. See Description.doc. Utricle hair bundles were purified from E20–21 chicks using the twist-off method (Gillespie & Hudspeth J Cell Biol 112, 625-640, 1991; Shin et al. Neuron 53, 371-386, 2007). NuPAGE LDS sample buffer (Invitrogen) with 50 mM dithiothreitol was added to a combined final volume of 80 µl per 100 utricles' worth of bundles; samples were heated to 70°C for 15 min. Epithelial proteins were similarly solubilized with sample buffer. Proteins were separated by running ~1 cm into a NuPAGE 4–12% Bis-Tris gel (1.5 mm x 10 well; one or two lanes per bundle sample); gels were rinsed with water, then stained with Imperial Protein Stain (Thermo Scientific). The 1 cm of gel with separated proteins was manually sliced into six pieces. Gel pieces were transferred to siliconized tubes and washed and destained in 200 µl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µl of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 µl 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 µl acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 µl 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 µl acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin (Sigma-Aldrich T6567 proteomics grade, from porcine pancreas, dimethylated) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess enzyme solution was removed and 20 µl 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in two 30 µl aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µl for MS analysis. For the gel slice immediately adjacent to the agarose, peptides were purified away from interfering polymers by SCX. A single experiment's worth of hair bundles or epithelium was analyzed by six LC-MS/MS runs, corresponding to the six gel pieces. The LC-MS/MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source, which was interfaced to a reversed-phase capillary column of 8 cm length x 75 µm internal diameter, self-packed with Phenomenex C18 Jupiter of 10 µm particle size. An extract aliquot (7.5 µl) was injected and peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µl/min over 1.2 hr. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent capability of the instrument, acquiring—in sequential scans—a single full scan mass spectrum in the Orbitrap detector at 60,000 resolution to determine peptide molecular weights, and 20 product-ion spectra in the ion trap to determine amino acid sequence. MaxQuant version 1.2.2.5 software was used for protein identification and quantitation. The default contaminants file associated with the MaxQuant download was edited to remove entries known to be present in hair bundles (e.g., actin) and to add additional impurities that entered the bundle-purification workflow (keratins, hemoglobins, egg white components). Mass spectrometry data were searched against Ensembl version 66 (released February, 2012) using Andromeda; the Ensembl FASTA file was edited by replacing several sequences with longer or full-length sequences, including actin gamma 1 (NP_001007825.1), actin beta (NP_990849.1), fascin 1 (NP_001171603), fascin 2 (NP_001171209), ATP synthase beta (NP_001026562.1), peptidylprolyl isomerase A (NP_001159798.1), calbindin 2 (NP_990647.1), PDZD7 (XP_003641537.1), espin (XP_417532.3), and CACNA2D2 (XP_427707.3). Protein identifications were reported with an FDR of 1%. If a set of peptides for a protein was identical to or completely contained within that of another protein, MaxQuant groups those proteins together ("redundant groups"); the entry with the largest number of peptides was used to identify the redundant group. Redundant groups that shared more than 20% of their identified peptides were further grouped in our analysis ("shared-peptide groups"); the entry with the greatest intensity associated with it was used to identify the shared-peptide group.

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Large-scale Proteomics Profiling of Peripheral Blood of DM1 patients identifies biomarkers for disease severity and functional capacity

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