Supplementary materials: Experimental section

High confidence and sensitivity four dimensional fractionation for human plasma proteome analysis

Authors: Millioni Renato1, Serena Tolin1,4, Gian Paolo Fadini1,4, Marco Falda2, Bas van Breukelen3, Paolo Tessari1, Giorgio Arrigoni2,4

Affiliations:

1 Department of Clinical and Experimental Medicine, University of Padua, Italy

2 Department of Biological Chemistry, University of Padua, Italy

3Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University and Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands

4 VIMM, Venetian Institute of Molecular Medicine, Padua, Italy

1 Sample preparation

The human blood sample was harvested in EDTA collection tubes from a healthy donor. After centrifugation at 1500 RCF for 10 min, plasma was separated from circulating cells and a cocktail of protease inhibitors (AEBSF, Sigma-Aldrich, St. Louis, USA) was added.

LAPs enrichment was performed on 1 ml of plasma using the ProteoMiner technology (BioRad, CA, USA), as previously described [1]. Enriched proteins were precipitated in four volumes of cold acetone (100%) overnight at −20°C. Samples were then centrifuged at 14000 RCF for 10 min and pellets were dissolved in 100 µl of 20 mM ammonium bicarbonate. Protein concentration was determined by a Lowry assay. Proteins (150µg) were reduced with 10 mM ditiotreitol for 1 hour and alkylated with 30 mM iodoacetamide for 1 hour at room temperature. Sample was then digested overnight at 37°C using sequencing grade modified trypsin (Promega, Madison, WI, USA) with an enzyme to substrate ratio of 1:20 (w/w).

2 Peptide fractionation

2.1 IPG-IEF

The peptide sample (150 µg) was diluted to 450 µl in 8 M urea and subjected to IPG-IEF on a strip of 24 cm 3-10NL IPG strip (Amersham, GE Healthcare) for about 100kVhr using the IPGphor (Amersham). After focusing, the strip was quickly washed in milliQ water to remove the mineral oil used to isolate the strip during IEF and dissected in eight fractions, named IEF(1-8). The strip washing and cutting were made within the shortest possible time (few minutes) because, in absence of voltage, the bands of focused peptides tend to diffuse. This phenomenon in not an issue when peptides are dissolved in a high viscosity solution such as 8M urea, where a band widening of a 100% occurs after two hours (Cargile BJ, 2005). Each IPG strip piece was transferred to an eppendorf tube and peptides were eluted with 200 µl of 0.1% formic acid for 15 min followed by 200 µl of acetonitrile/0.1% formic acid (1/1, v/v) for 15 min, then with 200 µl of acetonitrile containing 0.1% formic acid for 15 min. The eluted fractions were dried under vacuum, cleaned with C18 cartridges (SepPack, Waters) to remove the residual traces of oil, dried again under vacuum and finally resuspended in 100 µl of 0.1% formic acid.

2.2 SCX

Peptides separated by IPG-IEF fractionation were further fractionated using a cation exchange cartridge (AB Sciex, Toronto, Canada). Samples were diluted to 500 µl in an equilibration buffer composed of 5 mM KH2PO4, 25% acetonitrile, pH 3. Peptides were loaded onto the cartridge at 50 µl/min and washing was performed with 1 ml of equilibration buffer. Peptides were fractionated and stepwise eluted using each time 500 µl of elution buffer (5 mM KH2PO4, 25% acetonitrile, pH 3, with the addition of 50, 100, 200 and 350 mM KCl). Peptide fractions were dried under vacuum, resuspended in 1 ml of 0.1% formic acid and desalted with C18 cartridges (SepPack, Waters) according to the manufacturer instructions. Desalted samples were finally dried under vacuum, dissolved in 20 µl of 0.1% formic acid and analyzed by RP-LC-MS/MS.

2.3 Reversed-phase LC-MS/MS analyses.

The 32 peptide fractions obtained by IEF and SCX fractionations were analyzed by LC-MS/MS using a 6520 Q-TOF mass spectrometer coupled online with a 1200 series HPLC system through a Chip Cube Interface (Agilent Technologies, CA, USA). Five µl of each sample were loaded onto a C18 large capacity chip-column that integrates a 160 nl capacity trap-column, a RP column (75 µm×150 mm), connection capillaries, and nanospray emitter. Peptides were separated with a linear gradient of 0–50% of solvent B in 50 min at a flow rate of 0.5 µl/min. Solvent A was water/formic acid 0.1%, while solvent B was acetonitrile/formic acid 0.1%. Mass spectra were acquired in a data dependent mode: MS/MS spectra of the 3 most intense ions were acquired for each MS scan in the range of 350–2400 Da. Scan speed was set to 4 MS spectra/sec and 3 MS/MS spectra/sec. Capillary voltage was set to 1750 V and drying gas to 5 l/sec. Raw data files were processed into Mascot Generic Format (MGF) files with MassHunter Qualitative Analysis Software (Agilent Technologies) and analyzed using Proteome Discoverer Software (version 1.2, ThermoFisher Scientific, CA, USA) as described below.

3 Data analysis.

The MGF files were analyzed using Proteome Discoverer 1.2 (Thermo Fisher Scientific). The software was connected to a Sequest Search Engine version 28.0 (ThermoFisher Scientific) and each MS/MS spectrum was searched against the IPI Human database (version 24 February 2010, 86719 entries).

The searches were run using the following parameters: methionine oxidation, cystein carbamidomethylation modifications were respectively selected as variable and static modifications; enzyme specificity was set to Trypsin with up to 2 missed cleavages, peptide and fragment tolerance were set to 10 ppm and 0.05 Da respectively.Before the search, data were filtered to exclude MS/MS spectra containing less than 5 peaks and with a total ion count lower than 50. False Discovery Rates (FDR) of 0.5% and 0.1% were calculated by Proteome Discoverer based on the search against the corresponding randomized database. Identified peptides were classified as high (99%) and medium (95%) confidence, according to the corresponding FDR. For Sequest analysis, the following four settings were tested: 1) Xcorr≥2 for +2 tryptic peptides, ≥2.8 for +3 and +4 tryptic peptides for medium confidence identifications, Xcorr ≥1.9 for +2 tryptic peptides, ≥2.5 for +3 and +4 tryptic peptides for high confidence identifications; 2) Xcorr≥1.9 for +2 tryptic peptides, ≥2.7 for +3 and +4 tryptic peptides for medium confidence identifications, Xcorr ≥1.8 for +2 tryptic peptides, ≥2.4 for +3 and +4 tryptic peptides for high confidence identifications; 3) Xcorr≥1.8 for +2 tryptic peptides, ≥2.6 for +3 and +4 tryptic peptides for medium confidence identifications, Xcorr ≥1.7 for +2 tryptic peptides, ≥2.3 for +3 and +4 tryptic peptides for high confidence identifications; 4) Xcorr≥1.7 for +2 tryptic peptides, ≥2.5 for +3 and +4 tryptic peptides for medium confidence identifications, Xcorr ≥1.6 for +2 tryptic peptides, ≥2.4 for +3 and +4 tryptic peptides for high confidence identifications. Proteins were considered as positive hits if at least two peptides with medium confidence were identified per protein.

Peptides that could not be unequivocally attributed to a single protein were grouped into protein families to satisfy the principle of parsimony.Peptide pI values from peptides belonging from the same IEF fraction were calculated in batch using an algorithm developed by Gauci et al. [2] and peptides were considered positively identified if calculated theoretical pI corresponded to experimental pI with a ± 1 pH range cut off.

4. References

1.Millioni R, Tolin S, Puricelli L, Sbrignadello S, Fadini GP, Tessari P, Arrigoni G. High abundance proteins depletion vs low abundance proteins enrichment: comparison of methods to reduce the plasma proteome complexity. PLoS One 2011;6:e19603.

2.Gauci S, van Breukelen B, Lemeer SM, Krijgsveld J, Heck AJ. A versatile peptide pI calculator for phosphorylated and N-terminal acetylated peptides experimentally tested using peptide isoelectric focusing. Proteomics 2008;8:4898-4906.