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Protein Phosphorylation

Sigmascreen Selectivity
Product No: S 6940 (96-well, clear)
Product No: S 8815 (384-well, clear)

Selectivity of Binding on Streptavidin HC Plate
ptransxP1fig
Figure 1: MALDI-MS analysis of trypsin digested biotinylated lysozyme introduced to a Streptavidin HC plate from ion-exchange effluent of HPLC. A) supernatant of fraction 8 collected in plates, B) peptides eluted from Streptavidin HC plates using 10 mg/ml ACCA matrix in 70% acetonitrile, 0.1% formic acid. The peptides were introduced to the wells as fractions eluted from SCX ion-exchange HPLC in 25% acetonitrile 10 mM ammonium phosphate pH 3 with increasing amounts of 0.5 M ammonium chloride. The spectra show peptides eluted from a single well. The biotinylated peptides are designated in green.

Comparison of Streptavidin HC Plate to Monomeric Avidin Agarose
ptransxP2fig
Figure 2: (A) MALDI-MS analysis of trypsin digested biotinylated lysozyme prior to loading on either B) Streptavidin HC plates; or C) monomeric avidin agarose. Spectra B and C show purified biotinylated peptides eluted using 10 mg/ml ACCA matrix in 70% acetonitrile, 0.1% formic acid. Samples were loaded on the appropriate affinity matrix in 25% acetonitrile, 10 mM ammonium phosphate, 0.5 M ammonium chloride pH 3 at 2 nmole/ml. The biotinylated peptides are designated in green.

ACCA: a-cyano-4-hydroxycinnamic acid

Peptide Profile of Trypsin Digested Lysozyme
Designation From-To Sequence Mass (MH+)
T18 129-129 L 132.1809
T1 1-1 K 147.1956
T12 97-97 K 147.1956
T14 14-14 R 175.2091
T2 2-5 VFGR 478.5654
T10 69-73 TPGSR 517.5569
T15 115-116 CK 663.8523
T17 126-128 GCR 748.9172
T3 15-21 HGLDNYR 874.9210
T16 117-125 GTDVQAWIR 1046.1578
T3 5-13 CLLAAAMK 1250.5550
T9 62-68 WWCNDGR 1350.5273
T7 34-45 FESNFNTQATNR 1429.4715
T13 98-112 IVSDGNGMNAWVAWR 1676.8748
T6 22-33 GYSLGNVVVCAAK 1682.9631
T8 46-61 NTDGSTDYGILQINSR 1754.8303
T11 74-96 NLCNPCSALLSSDITASVNCAK 3579.2433

Table 1: Theoretical peptide fragments from trypsin digestion of lysozyme. The calculated mass values include the MW of the biotinylation reagent for the cysteine containing residues. The biotinylated peptides are designated in red.