Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis.
Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.
Anderegg
R J
RJ
Department of Physical and Structural Chemistry, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406.
Carr
S A
SA
Huang
I Y
IY
Hiipakka
R A
RA
Chang
C S
CS
Liao
S T
ST
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D011467:Prostate / Q000032:analysis*; D051381:Rats
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C032603:Sbp protein, rat; D004247:DNA