IL-17A driven hyperproliferation is a hallmark of psoriatic keratinocytes. The pathway analysis of shotgun proteomics data from human primary keratinocytes (HPKs) treated with IL-17A highlighted the role of increased levels of lactate dehydrogenase (LDH-A) and pyruvate kinase (PKM). We sought to validate these findings using multiple reaction monitoring (MRM) for LDH-A (6 peptides) and PKM (8 peptides). Moreover, speculating the role of the transcription factor HIF1a in regulation of LDH-A and PKM levels, we also probed these proteins upon inhibiting HIF1a activity with echinomycin in presence of IL-17A.

HPKs were treated with IL-17A (50ng/ml) and/or echinomycin (2.5nM) for 24 hours. Proteins were then extracted in urea (6M) and thio-urea (2M) buffer made in Tris-Cl. 25µg protein was then treated with dithiothreitol (DTT) and iodoacetamide (IAA) and digested in MS-grade trypsin (Promega, WI, USA). Peptides were desalted in C-18 silica based material, quantified. All transitions for unique peptides with 0 missed cleavages (precursor ion +2 charge, and product ion +1 charge) as predicted by the SkylineTM Daily software (Ver 22.2.1) were monitored. We first refined this list on a pool of IL-17A treated samples. 6 peptides of LDH-A (LVIITAGAR, NVNIFK, FIIPNVVK, QVVESAYEVIK, VTLTSEEEAR, SADTLWGIQK) and 8 peptides of PKM (LDIDSPPITAR, ITLDNAYMEK IYVDDGLISLQVK, GADFLVTEVENGGSLGSK, FGVEQDVDMVFASFIR, AGKPVICATQMLESMIK, LAPITSDPTEATAVGAVEASFK, and VNFAMNVGK) were finalized. 3 bio-replicates of each condition were used for the MRM experiment and results acquired were analysed on SkylineTM Daily. For the MRM sample runs, around 1.6 ug of peptides were injected onto Hypersil gold C18 column with an Ultimate 3000 UHPLC system. A binary buffer system (Buffer A = 0.1% Formic acid in water and Buffer B = 0.1% Formic acid in Acetonitrile) allowed for the separation of peptides that were fed directly into the Thermo TSQ Altis system for data acquisition. The cumulative peak areas for peptides were plot for each condition and hypothesis testing was performed using statistical analysis by Student’s t-test (paired). All the data and results have been made available here.

Clean peptides obtained from protein digests from the following samples: UT2: Untreated HPKs IL17A2: IL17A treated HPKs IL17AE2: Echinomycin and IL17A treated HPKs UT3: Untreated HPKs IL17A3: IL17A treated HPKs IL17AE3: Echinomycin and IL17A treated HPKs UT4: Untreated HPKs IL17A4: IL17A treated HPKs IL17AE4: Echinomycin and IL17A treated HPKs