IDENTIFICATION OF POSTTRANSLATIONAL MODIFICATIONS AND CDNA SEQUENCINGERRORS IN THE RAT S100 PROTEINS MRP8 AND 14 USING ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

MRP8 and 14 are 5100 proteins expressed by myeloid cells and are predi cted to have important functions in inflammation. The proteins were is olated from spleens from three rat strains. Electrospray ionization ma ss spectrometry indicated masses of 10149 +/- 2 Da for MRP8 and 13,069 +/- 2 Da for MRP14 compared to masses calculated from proteins derive d from their cDNA sequences of 10,211 and 13214 Ha, respectively, indi cating posttranslational modifications and/or errors in the derived pr otein sequences. Several endoprotease digest peptides did not correspo nd to any theoretical digest products after comparison of ESI masses w ith those derived from the theoretical digest. Both proteins were N-te rminally acetylated after deletion of the initiator Met, reducing the theoretical masses by 89 Da, A peptide with mass 28 Ha greater than th e theoretical was isolated from the Asp N digestion of MRP8. N-termina l sequencing indicated translated Val instead of the predicted Ala at position 72 of MRP8, A peptide 56 Da less than the theoretical was iso lated from the chymotryptic digestion of MRP14, and the carboxyamidome thylated form was N-terminally sequenced and found to have translated Ser instead of the predicted Arg at position 105, In addition, His(106 ) was methylated. The corrected theoretical masses, incorporating the posttranslational modifications and sequencing errors, are 10,149.4 an d 13,069.9 Da for MRP8 and 14, respectively, in good agreement with th e experimental masses. (C) 1998 Academic Press.