The test sample distributed this year, ABRF-94SEQ was chosen because previous ABRF sequencing samples had indicated that cysteine and tryptophan are often difficult residues for the majority of facilities to correctly identify. ABRF-94SEQ was a commercially available preparation of lactoferrin (ICN).
NH2-G-R-R-R-R-S-V-Q-W-C-A-V-S-Q-P-E-A-T-K-C-F-Q-W-Q-R-
This protein contains tryptophan at residues 9 and 23 and cysteine at positions 10 and 20. Two 50 pmol aliquots were sent to member laboratories along with methods for solution phase reduction of disulfide bonds (dithiothreitol or 2- mercaptoethanol in various denaturants), solution phase derivatization of cysteine residues (iodoacetic acid, iodoacetamide, vinyl pyridine, acrylamide, dimethylacrylamide, and N-isopropyliodoacetamide) and in situ derivatization of cysteine residues. A procedure for sample clean-up by centrifugation onto a PVDF membrane and a method for resolving tryptophan from the sequencing artifact diphenylurea on Applied Biosystems sequencers was also recommended. Members were encouraged to try at least one of the methods described and asked to return their completed surveys, sequence calls and chromatograms for the first 25 cycles to the committee for anonymous evaluation.
Seventy-eight responses were received and the results demonstrated a substantial improvement in the accuracy of positive assignments for Trp (95%) and Cys (67%) this year compared to ABRF-93SEQ (72% and 60%, respectively). Trp identification seemed problematic at later cycles of sequencing as evidenced by the much lower percent of positive correct calls for Trp23 versus Trp9 (82% and 45%, respectively). However, this was a remarkable improvement over ABRF-93SEQ, in which Trp2 and Trp7 were positively identified 49% and 46%, respectively. Cys10 and Cys20 were the most difficult residues to assign. Alkylation of Cys clearly improved the ability to identify cysteine residues, with overall accuracy of 88% versus 50% for non-alkylated samples. Positive cysteine identification (Cys10 [53%] and Cys20 [37%]) improved over the 1993 study, where Cys5 was positively identified at 20%. Although cysteine and tryptophan identification may be problematic, the data suggest that routine identification of these residues should be possible for the majority of facilities.
The members of the 1995 Protein Sequence Research Committee are continuing members Karen De Jongh (Cell Therapeutics, Inc., Seattle, WA), Jay Gambee (Shriners Hospital, Portland, OR), Greg Grant (Washington Univ., St. Louis, MO) (ad hoc), Barbara Merrill (Burroughs Wellcome, Research Triangle Park, NC), and John Rush (Harvard Medical School, Boston, MA), and new members Joseph Fernandez (Rockefeller Univ., New York, NY) and Kathy Stone (Yale Univ., New Haven, CT), who will replace Philip Andrews (Univ. Michigan, Ann Arbor, MI) and Sheenah Mische (Rockefeller Univ., New York, NY).
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