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Spatially resolved proteome profiling of 500 counts and charges between +2 and +7 were selected by the ion trap for MS/MS sequencing. The CID energy was set at 35% and ion activation time was 10 ms. The AGC target of ion trap was 10,000 and the maximum ion accumulation time was 150 ms. To reduce repeated sequencing, a dynamic exclusion with duration of 90 s and mass width of 50 ppm was used. Data Analysis. Raw data were analyzed using MaxQuant version 1.5.3.30.39 MS/MS spectra were searched against Solanum lycopersicum UniProt databases (35,871 total entries, 443 reviewed, downloaded Jan. 2018). Both peptides and proteins were filtered with a false discovery rate (FDR) of 0.01. Other search settings were as described previously,30 but the first and main search peptide tolerances were changed to 30 and 5
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Analytical Chemistry
ppm, respectively, to match the lower resolution LTQ Orbitrap instrument. Match Between Runs was activated when studying different cell types within pericarp. Labelfree protein quantification (LFQ) was used. Perseus (version 1.6.0.2) was used for qualitative and quantitative analysis.40 Potential contaminants and reverse sequences were filtered out. For quantitative cell-type-specific studies, datasets were grouped by cell type and protein intensities were normalized among different groups. The LFQ intensities for each sample were transformed with log2(x), and then filtered to contain ≥ 3 valid values out of 5 replicates in at least one group. Data quality was checked by pairwise correlation of all 15 samples to confirm no outliers. For principal component analysis (PCA), the missing values were imputed from a normal distribution with a width of 0.5 and downshift of 1.8.40 To identify significantly differentiated proteins among the three cell types, an analysis of variance (ANOVA) multiple sample test was performed with >4 fold differential accumulation (S0=2) and qvalue
