Nucleoid Protein Identification by SDS-PAGE and MS

The nucleoid proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to in-gel digestion with mass spectrometry (MS) grade modified trypsin (Promega). The peptides were extracted as described previously (26 (link)) and loaded onto a column (100-µm internal diameter, 15-cm length; L-Column, CERI) using a Paradigm MS4 HPLC pump (Michrom BioResources) and an HTC-PAL autosampler (CTC Analytics) and eluted over 26 min with a gradient of 5 to 45% (vol/vol) acetonitrile in 0.1% (vol/vol) formic acid. The eluted peptides were introduced directly into an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) with a flow rate of 500 nL-⋅-min⁻¹ and a spray voltage of 2.0 kV. The obtained spectra were compared to those in a protein database (Chlamydomonas genomic information 2.4) using the in-house MASCOT server (version 2.3, Matrix Science) (54 (link)). MASCOT search parameters were set as follows: threshold of the ion score cutoff, 0.05; peptide tolerance, 10 ppm; MS/MS tolerance, 0.5 Da; and peptide charge, +1, +2, or +3. The search was also set to allow one missed cleavage by trypsin, carboxymethylation modification of Cys residues, and variable oxidation of Met residues.

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Takusagawa M., Kobayashi Y., Fukao Y., Hidaka K., Endo M., Sugiyama H., Hamaji T., Kato Y., Miyakawa I., Misumi O., Shikanai T, & Nishimura Y. (2021). HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences of the United States of America, 118(20), e2021053118.

Publication 2021

MS) grade modified trypsin Paradigm MS4 HPLC pump HTC-PAL autosampler LTQ-Orbitrap XL mass spectrometer

Acetonitrile Chlamydomonas Cleavage Digestion Formic acid Genomic Hplc Mass spectrometry Mass spectrometry mass spectrometry Peptide Promega Proteins Sds page Tolerance Trypsin

Corresponding Organization : Kyoto University

Other organizations : Ibaraki University, Ritsumeikan University, Nara Institute of Science and Technology, The University of Tokyo, Yamaguchi University

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Variable analysis

independent variables

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) separation of nucleoid proteins
In-gel digestion of proteins with mass spectrometry (MS) grade modified trypsin

dependent variables

Peptides extracted from in-gel digestion
Peptides loaded onto column and eluted with acetonitrile gradient
Peptides introduced into LTQ-Orbitrap XL mass spectrometer
Obtained mass spectra compared to protein database using MASCOT server

control variables

Trypsin digestion parameters (one missed cleavage, carboxymethylation of Cys, oxidation of Met)
MASCOT search parameters (ion score cutoff, peptide tolerance, MS/MS tolerance, peptide charge)

positive controls

Comparison of obtained mass spectra to a protein database (Chlamydomonas genomic information 2.4)

negative controls

No negative controls were explicitly mentioned in the protocol.

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