**2.10 ZipTip**

After digestion and drying of peptide samples, some were desalted and purified by the use of ZipTipC18® pipette tips (Millipore, Billerica, MA, USA). Samples were diluted up to 10 µL with 0.1% TFA. A ZipTip was wet by loading 3x10 µL with 50% ACN and discarding the liquid. Ziptip was equilibrated by loading 3x10 µL of 0.1% TFA and discarding the fluid before peptide sample was carefully loaded into the ZipTip by pipetting. The ZipTip was washed with 5x10 µL of 0.1% TFA before peptides were eluted with 10 µL 50% ACN.

#### **2.11 Mass spectrometry**

Peptides obtained after digestion were mixed 1:1 with matrix, α-Cyano-4-hydroxycinnamic acid (CHCA, 0.02 mg/mL) or 2,5-dihydroxybenzoic acid (DHB, 0.02 mg/mL) in 70% ACN/0.3% TFA, and then spotted onto a stainless steel target plate. Analyses of peptide masses were performed using MALDI TOF-MS (Voyager DE PRO; Applied Biosystems) equipped with a 337 nm N2 laser operated in reflector mode with delayed extraction. Positive ionization, a delay time of 200 ns, and an accelerating voltage of 20 kV were used to collect spectra in the mass range of 600–3600 Da. Data processing of the spectra was performed in a Data Explorer TM Version 4.0 (Applied Biosystems). External mass calibration with a standard peptide mixture and internal calibration using known trypsin autolysis peaks (*m/z* 842.5100, 1045.5642, 2211.1046) were also performed prior to the database search. For tandem MS analysis, the digested peptides were dried and dissolved in 10 µL 0.1% TFA. The peptides were desalted and purified by using ZipTipC18® columns. Elution was acidified by the addition of 1% formic acid. About 2 µL of the sample was applied into a silver-coated glass capillary and analyzed by a hybrid (triple quadrupole-TOF) mass spectrometer (API Q-STAR Pulzer; Applied Biosystems) equipped with a nanoelectrospray ion source (MDS-Protana, Odense, Danmark) operated in the nanopositive mode. Data processing was performed with Analyst QS software (Applied Biosystems). Fragmentation spectra were interpreted manually.

#### **2.12 Database search**

Peptide masses (major peaks) in the spectra were submitted to database search. NCBI and Swiss-Prot were used with Aldente or MS-Fit as search engines. Restrictions were human species, mass tolerance >75 ppm in most of the searches, maximum one missed cleavage, and cysteine modification by carbamidomethylation. MS-Digest, MS product, and BLAST search was used for protein identification of the derived tags resulting from amino acid sequencing with MS/MS. In peptide mass fingerprinting, protein matches with p-values below 0.05 are used and with LC-MS/MS analyses an FDR < 1 % is considered significant.
