P 2000 laser puller

Proteins in the secreted and sheared fractions were identified with MS. Nano-electrospray ionization MS/MS (nanoESI-MS/MS) analyses were performed on an EASY-nLC II system (Thermo Fisher Scientific) connected to a TripleTOF 5600+ mass spectrometer (AB SCIEX) operated under Analyst TF 1.6.1 control. The trypsin-digested samples were suspended in 0.1% formic acid, injected, trapped and desalted on a precolumn. The peptides were eluted and separated on a 15 cm analytical column (75 μm i.d.), pulled in-house (P2000 laser puller, Sutter Instrument). Trap and analytical column were packed with ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH). Peptides were eluted from the analytical column at a flow rate of 250 nl/min using a 30 min gradient from 5 to 35% of solution B (0.1% formic acid, 100% acetonitrile). The collected MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.1 (AB SCIEX) and the “protein pilot MGF” parameters. The generated peak lists were searched using an in-house Mascot search engine (Matrix Science) against all C. acnes proteins in the UniProt database as well as against all tad plasmid-encoded proteins (the plasmid p09-9 was used). Search parameters were allowing one missed trypsin cleavage site and propionamide as a fixed modification with peptide tolerance and MS/MS tolerance set to 10 ppm and 0.2 Da, respectively.

SECCM probes were single-barrelled nanopipettes with approximately 400 nm aperture radius. The nanopipettes were fabricated from single-barrelled borosilicate capillaries (1.5 mm O.D and 0.86 mm I.D., BF150-86-7.5, Sutter Instrument, USA) using a P-2000 laser puller (Sutter Instrument, USA). Using a pipette filler (MicroFil MF34G-5, World Precision Instruments, USA) the nanopipette was filled with 20 mM HClO₄ electrolyte. A Pd-H₂ quasi reference counter electrode (QRCE) was inserted at the top end of the pipette; prior to this, a Palladium wire (0.25 mm diameter, 5 cm long, PD005130, Goodfellow, UK) was biased at −3 V vs. a Pt counter electrode in 20 mM HClO₄ solution for 15 min to yield the Pd–H₂ quasi reference electrode^{50 (link),51 (link)}. Pd–H₂ QRCE was calibrated against the standard calomel electrode (SCE) after the SECCM scan with a value of −191 mV, which corresponds to a potential of +50 mV vs Standard Hydrogen Electrode (SHE).

The carbon nanoelectrodes were fabricated according to the methods used for fabricating nanoscale tweezers (Supplementary Fig. 3). Briefly, the double-barrel theta quartz capillaries were first pulled by a two-line programme using a P-2000 laser puller (Sutter Instrument). Next, butane was passed through the pulled nanopipettes and heated, under an argon atmosphere to pyrolytically deposit carbon inside the pipette. After carbon deposition, the carbon nanoelectrodes consisted of two coplanar semi-elliptical electrodes encapsulated in quartz, which were separated by a quartz septum. All of the nanoelectrodes were fabricated immediately before experiments, unless noted otherwise, and stored in a sealed Petri dish until used, to minimise any contamination.