Procise 494

Edman sequencing of the antithrombin cleaved by enteropeptidase was performed under non-reducing conditions with Applied Biosystems Procise 494 equipment (Foster City, CA, USA). Because of the presence of a C-terminal disulfide bond between Cys247 and Cys430, cleavage at the reactive center loop of antithrombin is detected as the cleaved peptide remains bound17 (link)18 (link).

Trypsin-generated fragments from the affinity-purified Vip3Af1(WT) were electrophoretically separated in 12% SDS-PAGE. For Edman degradation analysis, proteins in the gel were transferred onto a PVDF membrane. Protein bands were then cut out and sent for Edman degradation. N-terminal amino acid sequencing was performed by using a Procise 494 (Applied Biosystems) at CIB-CSIC (Madrid, Spain).
For the peptide mass fingerprinting, protein bands were directly cut out from the gel and digested with trypsin. The peptide mass and sequence was determined by liquid chromatography and tandem mass spectrometry (LC-MS/MS) in a nanoESI qQTOF (5600 TripleTOF, ABSCIEX) at the proteomics facility of the SCSIE (Servei Central de Suport a la Investigació Experimental), at the University of Valencia (Valencia, Spain). The mass transitions were scanned first from 350–1250 m/z and then followed by a second scan from 100–1500 m/z. The peptides sequence identified were compared to the Vip3Af1(WT) protein sequence to match the region corresponding to each SDS-PAGE proteolytic band. Expected molecular weights were calculated using the online SIB Compute pI/Mw tool³⁸ .

Soluble protein extracts were prepared as described previously5 (link)61 (link) in a buffer containing 30 mM Tris, 4 mM EDTA (called TA buffer), 1 mM NAD, 20% glycerol and 40 μg mL⁻¹ protease inhibitor (Sigma). The protein concentration was assayed using the Bio-Rad (Hercules, CA, USA) reagent using bovine serum albumin as a standard.
Extracts were incubated for 15 min at 80 °C with 10% sodium dodecyl sulfate (SDS), 10 mM DTT, 20% glycerol, 0.2 M Tris and 0.05% bromothymol blue. Protein migration was performed on 12% polyacrylamide gel Mini-PROTEAN^® Tetra Cell (Biorad, Hercules, USA). Gels were either stained with Coomassie blue to determine any differential pattern at low and high CO₂ or the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, stained with red Ponceau, and used for N-terminal sequencing. LCIP63 was identified using a Synapt G1 mass spectrometer (Waters, Manchester, UK) coupled to a nano flow UPLC nanoAcquity (Waters). Spectra and protein search were processed by the PLGS 3.0.1 software (Waters) with the same search parameters as described below. N-terminal sequence determination was performed by Edman degradation using an automatic sequencer (Procise 494, Applied Biosystems). Mature sequence of LCIP63 was analysed using Hectar (http://webtools.sb-roscoff.fr/) to identify the location of the protein.

The GFP₁₇₂ and RFP_Strep liberated from the polyprotein precursor were purified by chromatography as described above, followed by separation on SDS-PAGE and transferred onto a PVDF membrane. The target band with the expected size was excised out after staining with Coomassie blue. The N-terminal sequencing was performed using Edman degradation on a Perkin Elmer Applied Biosystems Procise 494 protein/peptide sequencer coupled with an on-line Perkin Elmer Applied Biosystems Model 140C PTH Amino Acid Analyzer (Applied Biosystems, Calsbad, CA), performed by the Protein Core Facility at the Iowa State University.

The protein band that showed inhibition in the in-gel assay in Tricine-Native PAGE was further subjected to N-terminal amino acid sequencing using an ABI Procise 494 protein sequencer (Applied Biosystems), Iowa State University, US (Shekh and Roy, 2012 (link)).

N-terminal amino acid sequence analysis was performed as described previously (39 (link)). Briefly, collagenase digests after 20 h incubation were separated by gel filtration under aforementioned conditions, and tripeptide-containing fractions were collected. N-terminal sequence of the collected fractions was analyzed by a Procise 494 protein sequencer (Applied Biosystems) in pulsed-liquid mode.