Chemical modification and enzymatic fragmentation of peptides and

3.7.1 Reduction and alkylation of disulfide bonds

Reduction of disulfide bonds was performed by incubating the protein or synthetic peptide dissolved in 20 mM NH4HCO3 (pH 7.5) with a 10-fold molar excess of DTT per cysteine residue for 3 hours at 37 °C. Alkylation with iodoacetamide was then carried out by incubating the protein/peptide solution with a 2-fold molar excess of IAA over DTT for 3 hours at 25 °C in the dark. Synthetic peptides were then purified by RP-HPLC, while proteins were separated by 1D gel electrophoresis, followed by in-gel trypsin digestion and mass spectrometric analysis.

3.7.2 Proteolytic digestion of proteins using trypsin

Trypsin is a serine protease which cleaves at the carboxyl side of lysine and arginine residues, except when followed by proline. The trypsin used in this study (Promega, Mannheim, Germany) has been modified by reductive methylation. The reaction converts lysine residues to trypsin-resistant ε-N,N-dimethyllysine. The resulting alkylated trypsin has the same catalytic properties as the unmodified enzyme, but it is less susceptible to autolysis. This enables longer digestion times without producing unwanted peaks in the mass spectrum.

The proteins employed in extraction experiments were dissolved at 0.5-1 μg/μL in 20 mM NH4HCO3 (pH 7.5). In general, 50-120 μg protein were digested in a sample tube and aliquots of the resulting peptide mixture (equivalent to 20 μg protein) were employed in several extraction experiments. The digestion was carried

out by adding the required amount of trypsin to yield an enzyme to substrate ratio of 1:20-1:100 (w:w), followed by incubation for 2-18 hours at 37 °C. To establish the optimal digestion time for complete proteolysis, sample aliquots were removed at 1-3 hours time intervals and analyzed by 1D-SDS-PAGE and MALDI-MS. When the digestion was complete, the sample was frozen in liquid nitrogen and lyophilized. For on-column digestion (i.e. proteolytic excision), 20-40 μg protein and 0.5-2 μg trypsin (enzyme to substrate ratio 1:20-1:100, w:w) in PBS were used. The digestion was carried out for 3-18 hours at 37 °C.

In-gel digestion for protein sequence characterization was carried out by the following protocol. The protein spots (stained with Coomassie Brilliant Blue) were excised from the gel, cut into small pieces (~1 mm³) and placed into sample tubes. The gel pieces were then washed with water for 15 min., dehydrated by incubation for 30 min. in 60 % ACN and dried using a vacuum evaporator. The gel pieces were next rehydrated in 50 mM NH4HCO3 (pH 8) for 15 min. and dehydrated again with 60 % ACN. The rehydration-dehydration steps were repeated 2-3 times, until the gel pieces were destained. Afterwards, the gel pieces were dried in a vacuum evaporator and rehydrated for 45 min. on ice, with a cold solution containing 12.5 ng/μL trypsin in 50 mM NH4HCO3 (pH 8). The excess enzyme solution was then removed, the gel pieces were covered with 50 mM NH4HCO3 and incubated overnight at 37 °C. All solution volumes employed were adjusted to swell and cover the gel pieces completely, in general 100-200 μL being necessary. The peptide fragments produced were extracted by incubating the gel pieces with 60 % ACN, 0.1 % TFA in water for 1 hour at 25 °C.

The solution was collected and the procedure repeated 2 times. The solutions containing peptide fragments were pooled, lyophilized and analyzed by MALDI-MS or LC-MS/MS.

3.7.3 Proteolytic digestion of proteins using clostripain

Clostripain, also known as endoproteinase Arg-C, is a cysteine protease which cleaves preferentially C-terminally to arginine residues, even when arginine is followed by proline [218]. Cleavage also occurs after lysine residues, albeit with a

lower rate [219, 220]. The presence of calcium ions and reducing agents (such as DTT) is required for clostripain activity and the optimal pH is 7.4-7.8 [220].

Proteolytic digestion of galectins with clostripain was performed only in solution using the following protocol. A stock solution of clostripain was prepared by dissolving the lyophilized enzyme at 1 μg/μL in water. Aliquots of 20 μL enzyme solution were stored at -20 °C. Activation of clostripain was performed by mixing a stock solution aliquot with 20 μL 40 mM NH4HCO3, 2 mM CaCl2, 5 mM DTT (pH 7.6) and incubating for 3 hours at 37 °C. This procedure yielded an activated-enzyme solution of 0.5 μg/μL clostripain in 20 mM NH4HCO3, 1 mM CaCl2, 2.5 mM DTT (pH 7.6). The activated enzyme was stored at 4 °C and used within one week. The proteins to be digested were dissolved at 1 μg/μL in 20 mM NH4HCO3, 2.5 mM DTT (pH 7.6). 50-60 μL protein solution were then mixed with 2.5-6 μL active enzyme solution (yielding an enzyme to substrate ratio of 1:50-1:20) and incubated for 2-5 hours at 37 °C. The goal of the clostripain digestion was to achieve complete cleavage after arginine and minimal cleavage after lysine residues. For each protein, 1 μL aliquots were taken every 30-60 min. and analyzed by MALDI-MS, to verify the digestion yield and favored cleavage sites. When the digestion was considered complete, the reaction was quenched by freezing the sample in liquid nitrogen and subsequent liophylization.

3.7.4 Proteolytic digestion of proteins using chymotrypsin

Chymotrypsin is a serine protease which cleaves C-terminally to large hydrophobic amino acids (Phe, Tyr and Trp), except when they are followed by Pro.

Cleavage after other amino acid residues such as Met, Ile, Ser, Thr, Val, His, Gly and Ala is also possible, but with lower rates [221]. Chymotrypsin was employed for primary sequence characterization and extraction experiments of galectin-1 and -3.

The stock chymotrypsin solution was prepared by dissolving the enzyme at 1 μg/μL in 1 mM HCl, 2 mM CaCl2 and kept at -20 °C. The proteins to be analyzed were dissolved at 1 μg/μL in 20 mM NH4HCO3 (pH 7.5) and digested for 6-18 hours at 37 °C, using an enzyme to substrate ratio of 1:20.