Identification of posttranslational modifications and cDNA sequencing errors in the rat S100 proteins MRP8 and 14 using electrospray ionization mass spectrometry.
MRP8 and 14 are S100 proteins expressed by myeloid cells and are predicted to have important functions in inflammation. The proteins were isolated from spleens from three rat strains. Electrospray ionization mass spectrometry indicated masses of 10,149 +/- 2 Da for MRP8 and 13,069 +/- 2 Da for MRP14 compared to masses calculated from proteins derived from their cDNA sequences of 10,211 and 13,214 Da, respectively, indicating posttranslational modifications and/or errors in the derived protein sequences. Several endoprotease digest peptides did not correspond to any theoretical digest products after comparison of ESI masses with those derived from the theoretical digest. Both proteins were N-terminally acetylated after deletion of the initiator Met, reducing the theoretical masses by 89 Da. A peptide with mass 28 Da greater than the theoretical was isolated from the Asp N digestion of MRP8. N-terminal sequencing indicated translated Val instead of the predicted Ala at position 72 of MRP8. A peptide 56 Da less than the theoretical was isolated from the chymotryptic digestion of MRP14, and the carboxyamidomethylated form was N-terminally sequenced and found to have translated Ser instead of the predicted Arg at position 105. In addition, His106 was methylated. The corrected theoretical masses, incorporating the posttranslational modifications and sequencing errors, are 10,149.4 and 13,069.9 Da for MRP8 and 14, respectively, in good agreement with the experimental masses.
Raftery
M J
MJ
Cytokine Research, School of Pathology, University of New South Wales, Kensington, Australia. m.raftery@unsw.edu.au
Geczy
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040501:Calgranulin A; D040502:Calgranulin B; D018076:DNA, Complementary; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats; D011916:Rats, Inbred F344; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid
D000943:Antigens, Differentiation; D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D040502:Calgranulin B; D018076:DNA, Complementary