Fig. 2: RgpB proteolytically modifies the intracellular neutrophil proteome.

Live human neutrophils were incubated with active 300 nM RgpB (gLN) for buffer alone (Buffer) and subject to terminal amine isotopic labeling of substrates (TAILS) mass spectrometry (TAILS-MS). A Schematic depicting TAILS-MS workflow (generated using Biorender.com). Briefly, after blocking primary amines (not shown), samples underwent isotopic labeling with heavy (deuterated) or light formaldehyde and digestion with trypsin. After trypsin digestion, a fraction of each sample was subject to pre-enrichment TAILS (shotgun analysis or pre-TAILS). The rest underwent removal of tryptic N-terminal peptides using a high molecular weight dendritic polyglycerol aldehyde polymer, leaving the naturally blocked or labeled mature and neo-N-termini unbound via negative selection (flow-through). TAILS peptides were recovered by size exclusion filtration and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). B The numbers of unique and shared peptides between TAILS and preTAILS analysis are shown. C The numbers of statistically changing peptides using an interquartile boxplot analysis between the pre-TAILS samples are shown. D Left: Distribution of N-terminal peptides in the TAILS enrichment. Middle, statistically changing peptides using an interquartile boxplot analysis. Right, Distribution of post-translational peptide modifications as analyzed using TopFINDER. For a complete list of peptides in figures (B, C), also see Supplementary Table 1. E Left, peptide sequence profiles of significantly elevated neo-N-terminal peptides in RgpB treated neutrophils identified in the TAILS analysis using IceLogo. Right, Cleavage sites identified as RgpB-treated neutrophils are depicted as heatmaps from P6 to P6′ residues. Green: Upregulated. Red: Downregulated. F Left, peptide sequence profiles of significantly elevated neo-N-terminal peptides in untreated (buffer) neutrophils identified in the TAILS analysis using IceLogo. Significantly (p < 0.05) overrepresented amino acids are shown above the x-axis, while the underrepresented residues are shown below the x-axis. Statistical analysis was determined by a two-tailed unpaired Student’s t-test and adjusted for multiple comparisons. G Metascape analysis of the TAILS data of different pathways between buffer- and RgpB-treated neutrophils is shown. H STRING-db analysis of RgpB cleaved substrates is shown. An enrichment was detected for neutrophil degranulation (blue), signaling by interleukins (red) and antimicrobial peptides (green). The dotted circle shows an enrichment of neutrophil azurophilic granule proteins (cathepsin G (CTSG); neutrophil elastase (ELANE); myeloperoxidase (MPO) and azurocidin (AZU1)) as RgpB substrates.