Published April 16, 2020 | Version v1
Dataset Open

Evaluation SIHUMI dataset: A sectioning and database enrichment approach for improved peptide spectrum matching in large, genome-guided protein sequence databases

Authors/Creators

  • 1. University of Minnesota
  • 2. University of Washington

Description

Dataset for evaluation of database sectioning method for generating an enriched database for mass-spectrometry-based proteomic approaches using large databases. Our evaluation demonstrates that this method helps to increase the sensitivity of PSMs while maintaining acceptable FDR statistics. This dataset was acquired from the protein samples containing proteins from eight microorganisms (Anaerostipes caccae, Bacteroides thetaiotaomicron, Bifidobacterium longum, Blautia producta, Clostridium butyricum, Clostridium ramosum, Escherichia coli, Lactobacillus plantarum) that were grown in a bioreactor. The MS-data for this dataset was acquired by Dr. Hettich's group at Oak Ridge National Laboratory, and it was made available by Dr. Nico Jehmlich from Helmholtz Center for Environmental Research through the 3rd International Metaproteome Symposium (https://www.ufz.de/index.php?en=44639). 

Notes

Sample: SIHUMI

Protein extraction: Bead-beating in 4% SDS, 100 mM Tris-HCl, Chloroform-methanol extraction, resolubilized in 4% sodium deoxycholate (SDC) in 100 mM ammonium bicarbonate (ABC) buffer for digestion
Replicate: single extraction
Protein quantification: BCA assay
Protein concentration [µg/µl]: Peptide concentration after diegestion 1.783µg\µL

Protein fractionation:
Digest type: On-filter (10kDa MWCO spin filters (Vivaspin 500, Sartorius))
Digested protein amount [µg]: 250 µg
Reduction: 10mM DTT, 10 min, 85°C
Alkylation: 30mM Iodacetamide, RT, 20min, in the dark
Enzyme (enzym:protein-ratio): trypsin 1:75

Lyophilization: yes

LC
Sample volume [µl]: 2µL
Loaded peptide approx. [µg]: 2µg
Trap column: direct loaded on to the analytical column
Separation column: 50 cm × 75 μm i.d. column packed with 1.7 μm Kinetex particle size Kinetex C18
Flow rate nano pump [nl/min]: 250
Length of gradient [min]: 240 min

MS
Instrument: Thermo Fischer Q Exactive Plus
Measurement modus: DDA-Top 10
Mass accuracy MS1: 5ppm
Mass accuracy MS2: 0.02 Da
Raw data format: raw
Raw data file(s): ORNL_Easy_nLC1200_contest2_C1_2ug_UHPLC_gradient240min_ac50cm.raw

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