Figure 1. Distribution of proteins identified according to the subfractionation workflow.

(a) Workflow of the proteomic experiment. The preparation of the five different subcellular fractions is exemplified with the most crucial steps. The starting point for all proteomic samples is the bacterial batch cell culture. The first crossroads is the separation between complete cells for the BEF (biotinylation-enriched fraction) aiming at the surface-exposed proteins, the cell culture medium for the extracellular proteins and cells for the cytosolic/membrane fractions. ¹⁴N/¹⁵N mixing depicts the stage in sample preparation, where mixing of the labelled pool and the unlabelled samples occurred. The earliest time point in experimental setup is chosen to minimize introduced effects by the labelling procedure to the quantitative data. Proteomic analyses (GeLC-MS and LC-MS) were performed with high-resolution and high mass accuracy mass spectrometry (2,142 total identified proteins with an FPR (false-positive rate) of 0.11% and 1.86 p.p.m. mass accuracy). (b) Distribution of proteins identified with a predicted subcellular localization in the different subfractions analysed. The area of the circles is proportional to number of identified proteins. (c) VENN plot of protein identifications in the different designated subcellular fractions. Numbers for the membrane fraction are summarized from EMF and MSF. Prediction of subcellular localization is according to Zhou et al.52 Loci of localization assigned were as follows: intracellular; integral membrane proteins (multitransmembrane, multitransmembrane (lipid-modified N termini)); membrane-associated proteins (lipid anchored, LPxTG cell wall anchored, N-terminally anchored (no cleavage site), N-terminally anchored (with cleavage site), C-terminally anchored (with cleavage site), intracellular/TMH start after 60); secreted (secreted via minor pathways (bacteriocin) (no cleavage site), secretory (released) (with cleavage site)).