Project PXD002606

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Evening Complex associated proteins in Arabidopsis by AP-MS


To determine how clock components are integrated with cellular pathways, affinity purification and mass spectrometry (AP-MS) were used to identify proteins that co-precipitate with the evening complex, which is critical regulator of clock, growth, light and flowering pathways.

Sample Processing Protocol

Plant extracts were incubated with anti-FLAG antibody coupled to magnetic beads. Captured proteins were eluted using 3xFLAG peptide then combined and depleted using Talon magnetic beads. The proteins binding to the magnetic beads were reduced, alkylated then digested with trypsin at 37°C overnight. The digest was dissolved in 5% ACN/0.1% formic acid and 5 µL were injected to the LC-MS/MS system. The LC-MS/MS was carried out on a LTQ-Orbitrap Velos Pro (ThermoFisher Scientific, Waltham, MA) coupled with a U3000 RSLCnano HPLC (ThermoFisher Scientific). The protein digests were first loaded onto a C18 trap column (PepMap100, 300 µm ID  5 mm, 5 µm particle size, 100 Å; ThermoFisher Scientific) at a flow rate of 5 µL/min. Peptide separation was carried out on a C18 column (Acclaim PepMap RSLC, 15 cm × 75 μm nanoViper™, C18, 2 μm, 100 Å, ThermoFisher Scientific) at a flow rate of 0.26 L/min and the following gradient: Time = 0-4 min, 2 % B isocratic; 4-8 min, 2-10% B; 8-83 min, 10-25 % B; 83-97 min, 25-50 % B; 97-105 min, 50-98%. Mobile phase A, 0.1 % formic acid; mobile phase B, 0.1 % formic acid in 80:20 acetonitrile:water. The Orbitrap mass analyzer was operated in positive ionization mode using collision induced dissociation (CID) to fragment the HPLC separated peptides. The mass range for the MS survey scan done using the FTMS was 300 to 2000 m/z with resolution set to 60,000 @ 400 m/z and the automatic gain control (AGC) target set to 1,000,000 ions with a maximum fill time of 10 ms. The 20 most intense signals in the survey scans were selected and fragmented in the ion trap using an isolation window of 1.5 m/z, an AGC target value of 10,000 ions, a maximum fill time of 100 ms, a normalized collision energy of 35 and activation time of 30 ms.

Data Processing Protocol

The mass spectra data were extracted by Proteome Discoverer (ThermoFisher Scientific; v.1.4) and converted into mgf. The database search was done using Mascot (Matrix Science, London, UK; v.2.5.0). Mascot was set up to search the cRAP database ( and the TAIR database assuming the digestion enzyme trypsin and 2 missed cleavages. Mascot was searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 15 PPM. Deamidated of asparagine and glutamine, oxidation of methionine and carbamidomethyl of cysteine were specified in Mascot as variable modifications. Scaffold (Proteome Software Inc., Portland, OR; v.4.4.3) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm with Scaffold delta-mass correction. The Scaffold Local FDR algorithm was used and only peptides probabilities with FDR<1% were used for further analysis. Protein identifications were accepted if they could be established at greater than 99.0% probability. The Normalized Spectral Abundance Factor (NSAF) was selected for the quantitative measurement in Scaffold to estimate the protein abundance of individual proteins in samples.


Alvarez Sophie, Proteomics and Mass Spectrometry Facility
Dmitri Nusinow, Danforth Plant Science Center, St Louis, MO ( lab head )

Submission Date


Publication Date



    Huang H, Alvarez S, Bindbeutel R, Shen Z, Naldrett MJ, Evans BS, Briggs SP, Hicks LM, Kay SA, Nusinow DA. Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry. Mol Cell Proteomics. 2016 Jan;15(1):201-17 PubMed: 26545401


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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 58499 no assay title provided (mzIdentML) 275 1779 700 1261 1261
2 58498 no assay title provided (mzIdentML) 201 939 311 675 675
3 58510 no assay title provided (mzIdentML) 87 1087 277 905 903
4 58512 no assay title provided (mzIdentML) 79 948 259 812 808
5 58511 no assay title provided (mzIdentML) 59 1154 236 985 981
6 58514 no assay title provided (mzIdentML) 39 1069 231 931 930
7 58508 no assay title provided (mzIdentML) 112 1127 323 971 968
8 58513 no assay title provided (mzIdentML) 39 1037 237 914 913
9 58509 no assay title provided (mzIdentML) 103 1035 293 876 875
10 58506 no assay title provided (mzIdentML) 130 1253 423 1010 1010