Ultraflex 2

The peptide digest extracted from the gel piece (1 μ l) was premixed with same volume of the matrix and spotted on a matrix-assisted laser desorption ionization (MALDI) plate. The Peptide mass fingerprint (PMF) was analyzed using MALDI TOF- mass spectrometer in the reflector mode (Ultraflex II, Bruker Daltonics, Germany). Swiss-Prot database using MASCOT search engine (Matrix Science, London, United Kingdom) with a peptide mass tolerance of 100 ppm was used to search the data generated.

Dried peptides were concentrated and de-salted using Zip-Tips C18 (microbed) (Millipore, Billerica, MA) according to the manufacturer’s instructions.
After elution from the Zip-Tip 0.5 µL of the de-salted peptides were spotted with α-cyano-4-hydroxycinnamic acid onto a ground steel MALDI target plate (Bruker Daltonics, Bremen, Germany). MALDI-TOF/TOF mass spectrometry (Ultraflex II, Bruker Daltonics, Bremen, Germany) was used for spectra acquisition in MS and MS/MS modes. Spectra processing and peak annotation were carried out using FlexAnalysis 3.0 and Biotools 3.2 (both Bruker Daltonics, Bremen, Germany).
Processed spectra were searched via an in-house Mascot server version 2.4.1 (Matrix Science, Boston, MA) and the software ProteinScape 2.1 (Bruker Daltonics, Bremen, Germany) in the UniProt database of sus scrofa using the following search parameters: global modification carbamidomethylation on cysteine; variable modifications oxidation on methionine; deamidation on asparagine and glutamine as well as formation of pyroglutamic acid; enzyme specificity trypsin; charge state z = 1; MS tolerance 100 ppm; MS/MS tolerance 1 Da; two missed cleavages allowed; significance threshold p < 0.05.

The peptides were analyzed using an ULTRAFLEX II (Bruker Daltonics, Bremen, Germany) MALDI-TOF/TOF mass spectrometer in positive ion reflector mode. 0.4 µL of the sample was mixed with 0.4 µL alpha-cyano-4-hydroxycinnamic acid (Bruker Daltonics) matrix solution (10 mg mL⁻¹ in 0.1% trifluoroacetic acid/acetonitrile) in a 1:1 ratio and spotted onto a MALDI plate. For some peptides, the samples were purified using a µ-C18 ZipTip (Millipore, Billerica, MA, USA) and directly spotted onto the MALDI plate with 0.6 µL alpha-cyano-4-hydroxycinnamic acid. The mass spectra were internally calculated from the raw mass spectra using the SNAP algorithm in FlexAnalysis 2.4 (Bruker Daltonics). Expected masses were calculated using the PeptideSynthetics Peptide mass calculator [42 ].

10 μL, equivalent to 11 μg proteins, of desalted serum (Merck Millipore ZipTip, Milano, Italy) and 10 μL of dialyzed urine were mixed with 10 μL of saturated HCCA (α-cyano-4-hydroxycinnamic acid) prepared in 0.1% trifluoroacetic acid (TFA) and acetonitrinile (ACN) (2:1 v/v). 1 μL of this mixture was spotted on a ground steel MALDI-TOF/MS target. Crystallization was always performed at constant humidity and temperature ranges during all the experimental sessions. MALDI-TOF/MS measurements were taken using an Ultraflex II instrument (Bruker Daltonics, Bremen, Germany) operating in conditions described elsewhere [18 (link)]. The mass to charge (m/z) ratio ranging from 1000 to 4000 was analysed. Spectra processing was performed as described in detail in the Additional file 1: Materials and methods section.

Following the selection of the spots of interest, i.e., spots detected in all three replicates, the protein spots corresponding to the Western blots were excised from the gel, destained, and subjected to overnight trypsin digestion (0.01 µg/µL) (Promega; Mannheim, Germany) as previously described [44 (link)]. The digested precipitates were reconstituted in 3.5 µL 5% acetonitrile in 0.1% TFA (trifluoroacetic acid; Merck; Darmstadt, Germany). The reconstituted precipitates were then spotted on to target plates for matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) on a Bruker Ultraflex II instrument (Bruker Daltonik; Bremen, Germany) using HCCA (α-cyano-4-hydroxycinnamic acid; Sigma-Aldrich; Steinheim, Germany) as matrix. A database search was conducted against all entries using the MS/MS ion search mode (MASCOT, http://www.matrixscience.com) as previously described [36 (link)]. Protein identification was considered valid if more than two peptides matched and the MOWSE score was significant (p < 0.05).

IR spectra of the samples were recorded in potassium bromide pellets using a Nicolet 380 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). ¹H NMR spectra were registered on Bruker CXP 200, Chemical shifts in ¹H spectra were measured relative to the signal of (CH₃)₄Si. Micro photo images were processed using the MCview (Lomo Microsystems, St-Petersburg, Russia) software package via Lomo MSP-2-2SD stereoscopic microscope equipped with an MC-5 electronic camera (Lomo Microsystems, Russia). MALDI spectra were recorded on an Ultraflex II mass spectrometer (Bruker, Karlsruhe, Germany) with an accelerating voltage of 25 kV with Nd: YAG laser (355 nm) desorption from a DHB matrix.

Gold AB plates were loaded into the instrument using the MTB AB adapter (Bruker). MALDI-ToF mass spectra (1500 shots/spectrum) were recorded on a Bruker Ultraflex II instrument with a Smartbeam I laser in positive reflector ion mode. The instrument was calibrated between m/z 700–3500 using a solution of peptide calibration mix II (Bruker Daltonics, Bremen). Data were analysed and normalised using FlexAnalysis version 3.0 (Bruker).