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  • Annotation of proteins identified by MS

    2018-11-07

    Annotation of proteins identified by MS/MS analysis was performed by querying a variety of databases (genomic and transcriptomic) using BLAST (BLASTp, tBLASTn and PSI-BLAST) algorithms [5]. Results are compiled in Supplemental Table S2 and Fasta files containing protein sequences [1]. These files are: (1) MS-identified mtDNA-encoded proteins; (2) MS-identified nuDNA-encoded mitochondrial proteins; (3) MS-identified putative nuDNA-encoded non-mitochondrial proteins; (4) putative mitochondrial proteins identified by bioinformatic means (in silico) only. Some of these data have previously been published [6–9]. A Mascot semi-tryptic peptide search was employed to detect putative mature protein N-termini, essentially as described [10]. Briefly, semi-tryptic peptides with ion scores surpassing the Mascot identity threshold and lacking an N-terminal Arg or Lys tryptic cleavage site were considered. If a semi-tryptic peptide lacking an N-terminal Arg or Lys was located in the N-terminal region of the inferred protein sequence (usually the first 50–60 amino acids) and not located in a region of the protein conserved in other species, it was considered to be the putative mature protein N-terminus [2]. OrthoMCL [11] was used to identify orthologs/co-orthologs of A. castellanii proteins in the predicted cellular proteomes of Arabidopsis thaliana (35,386 sequences from TAIR 10 protein dataset), Homo sapiens (20,270 sequences from UniProt), Saccharomyces cerevisiae (6,572 sequences from Saccharomyces Genome Database), and Tetrahymena thermophila (27,054 sequences from NCBI nr database), all of which have been examined intensively at the level of the mitochondrial proteome. For A. castellanii protein sequences, we used a six-frame translation of RNA-seq data supplemented with corrected versions of each mitochondrial protein identified in our analysis. OrthoMCL was run according to the recommended parameters, with an E-value threshold of 1e−5. Data are compiled in Supplemental Table S3.
    Specifications table
    The data is directly available in this article and related to de Luna-Valdez et al. (2014) . Experimental design Total protein was extracted from 16-days old mutant and 8-days old wild-type plants by triplicate. 2-D PAGE gel images were generated and compared in order to discover reliable (T-test P<0.01) spots with abundance shift of at least ±2-fold. Protein identification was performed using MALDI-TOF Mass spectrometry.
    Acknowledgments The authors thank Patricia Jarillo for the technical support, Alma Lidia Martínez, Juan Manuel Hurtado, Roberto Rodríguez Bahena and Arturo Ocádiz for computer support, Paul Gaytan and Eugenio López for oligonucleotide synthesis and Guadalupe Zavala Padilla (UME, Instituto de Biotecnolgía, UNAM) and Fernando García Hernández (UM, Instituto de Fisiología Celular, UNAM) for chloroplast Kinase Inhibitor Library microscopy analysis. This work was supported by UNAM-DGAPA-PAPIIT (Grants IN217111 to AGG and IN208211 to PL) and CONACYT-México (CB-129266 to AGG and 127546 to PL).
    Data, experimental design, materials and methods
    Establishment of fibroblast cultures from fresh surgical specimen We established two pairs of colon cancer-associated fibroblast (CAF) and normal fibroblast (NF) cultures. The fresh colorectal cancer tissues and adjacent normal colonic tissues (at least 5cm away from the loci of cancerous tissue) from two patients with colon ulcerated adenocarcinoma were collected during surgery at the Zhongshan Hospital of Fudan University according to the procedure described in the Journal of Proteomics paper [1]. The clinic and pathological data of the two patients are available in Supplementary Table 1.
    Proteomic analysis of the colonic CAF and NF secreted proteins (SPs) Fifty micro gram of the SP extracted from the 1031_NF and 1031_CAF conditioned medium (CM) were separated using 10% SDS-PAGE. Equal amount of sample was loaded in triplicate. Two gel lanes of each sample were cut into 10 slices separately and subjected to in-gel tryptic digestion according to an optimized procedure [2]. The digested peptides were analyzed using Synapt G1 mass spectrometer as described previously [3]. Briefly, the peptide mixture from each gel slice was first loaded onto a reverse phase (RP) trap column (C18, 5μm, 180μm×20-mm Symmetry C18 nanoAcquity column, Waters) for on-line desalting at a flow rate of 10μL/min. The peptides were then eluted from the trap column into an analytical fused silica nanoAcquity UPLC column of 75μm×200-mm inner diameter packed with C18 of 1.7-μm pore diameter stationary phase (Waters). The column oven temperature was maintained at 40°C. Mobile phase A contains 0.1% formic acid (FA) in water, while mobile phase B was 100% acetonitrile (ACN)/0.1% FA. The mobile phases were delivered using a nanoAcquity UPLC system (Waters) at a flow rate of 250nL/min. The peptides were separated with a linear gradient of 3–15% mobile phase B (5min), 15–35% B (65min), 35–98% B (5min), 98% B (5min), and 98–3% B (10min). The peptides were ionized at the end of a 10-μm-inner diameter PicoTip nanospray emitter (New Objective) which was connected to the end of the analytical column. A voltage of 3200V was applied to the emitter for a steady spray. The ions got into a Synapt G1 mass spectrometer (Waters) via a Nano electrospray ionization (ESI) source. The source-inside temperature was maintained at 100°C. During the data acquisition, a reference sprayer using 320fmol/μL lock mass compound [Glu1]-fibrinopeptide B (GFB) (Sigma) was sampled with a frequency of 30s at a flow rate of 200nL/min. The precision of the mass data was calibrated using the doubly charged monoisotopic ion of GFB. The MSE data was acquired using alternating low-energy collision at 6eV for survey scan and high-energy collision ramped from 15 to 55eV for tandem MS. The spectral acquisition time in each mode of 1.2s and a full cycle was 2.44s. The mass range of the survey scan was set at m/z 100 to 1800.