2.5. Protein identification Protein

2.5. Protein identification
Proteins were either identified after SDSPAGEby in-gel or by on-tissue (“on polymer”) digestion. For the first,gel bands were excised with a clean scalpel and destained using 100 mM 〖Na〗_2 S_2 O_3 and 30 mM K_4Fe〖(CN)〗_6•3H_2O (1:1,v/v).Gel pieces were treated with ACN and rehydrated with 100 mM 〖NH〗_4 〖HCO〗_3. After reduction (10mM DTT in 100 mM 〖NH〗_4 〖HCO〗_3) and alkylation (50 mM iodoacetamide in 100 mM 〖NH〗_4 〖HCO〗_3) the gel pieces were dried in a vacuum centrifuge and rehydrated in approx.10 µL 50 mM 〖NH〗_4 〖HCO〗_3 (pH 8.5) containing 5% ACN and 125 ngtrypsin (porcine,proteomics grade,Roche,Switzerland). Digestion was carried out for 24 hat 37 ◦C. Peptides were extracted with 50 mM 〖NH〗_4 〖HCO〗_3/CAN (1/1,v/v) and two times with uH2O/CAN containing 0.1% TFA (1/1,v/v), for 15 min each. All extracts of one respective spot were pooled and dried in a vacuum centrifuge.After reconstitution in 0.1% TFA peptides were desalted using C_18 ZipTips (Millipore,USA) and eluted with 5 mg/mL HCCA prepared in ACN/0.1% TFA(50/50,v/v) in the final step.Peptide mass fingerprinting(PMF) and sequence tag analysis were carried out on a MALDI-TOF/RTOF instrument (UltrafleXtreme). For“onpolymer”enzymatic treatment,the ChIP-1000 was used for precise trypsin deposition at a standard concentration of 3ng/ µm^2 containing 1% Rapigest (Waters,USA) dissolved in 50 mM 〖NH〗_4 〖HCO〗_3 (pH 7.5) containing 5% ACN.Trypsin solution was applied at a lateral resolution of 100 µm covering the complete sample area.For digestion samples were stored at 37 ◦C for 24 h in saturated atmosphere (ethanol/water,50/50,v/v, 80% relative humidity).After incubation samples were carefully washed with uH2O to remove salts,delocalized peptides not adsorbed to thepolymer and autolytic fragments from trypsin.Samples were vacuum dried for 15min and HCCA was applied afterwards as described. For all enzymatic digestion data,autolytic tryptic products, keratin and blank artifacts were assigned and removed before database search (SWISSPROT,December 2013) using Mascot with the following parameters:taxonomy Homo sapiens, monoisotopic mass values,peptide mass tolerance of ±0.3 Da (for PMF and PSD experiments),2 missed cleavages,afixed modification carboxyamidomethylation and methionine oxidations set as variable modification.