Abstract
The primary structure of rat heart muscle fatty acid-binding protein was investigated by liquid secondary ion mass spectrometry. The protein was digested with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and the resulting peptides were separated by reverse phase high performance liquid chromatography. The masses of the protonated molecular ions (MH+) of the tryptic, chymotryptic, and S. aureus protease peptides were determined by liquid secondary ion mass spectrometry analysis using 20-500 pmol of material. From the tryptic digest, two peptides with MH+ 1036 and 861 were initially found that did not match the published primary sequence (Sacchettini, J. C., Meininger, T. A., Lowe, J. B., Gordon, J. I., and Banaszak, L. J. (1987) J. Biol. Chem. 262, 5428-5430). The amino acid sequences of these two peptides were determined by a combination of mass spectrometry, B/E-linked scanning, and high performance tandem mass spectrometric techniques to be: (Formula: see text). These new data require that corrections be made to the previously published sequence, involving residues 1-4 and 51-52. The corrected amino sequence for rat m-FABP reveals greater homology with myelin P2, mouse adipocyte p422 protein, and intestinal fatty acid-binding protein than was previously demonstrated.
Highlights
The cellular retinol and retinoic acid-binding proteins [4, 8, 9]
5430).The amino acid sequences of these two peptides were determined by a combination of mass spectrometry, B/E-linked scanning, and high performance tan- Materials-Trypsin (L-1-tosylamido-2-phenylethyclhloromethyl dem mass spectrometric techniques to be: ketone-treated) and a-chymotrypsin were purchased from Sigma,and
From the trypticdigest of rat heart FABP(see Fig. lA), 17 peptide containing fractions were separated by HPLC and analyzed by liquid secondary ion mass spectrometry (LSIMS)
Summary
50s double-focusingmass spectrometer operating at a mass resolution of 2500. A Cs' ion beam of 8 keV was used for this instrument with an 8-keV accelerating voltage previously described [16].Peptide samples were generally in the 100-500-pmol range, and a scan was taken at 300 s/decade with signal detection using an electron multiplier and. For the peptide with MH' = 861, a B/E scan was taken with no attenuation of the sample ion beam to detect metastable decomposition products. A MS/MS experiment was carried out using a high performance tandem mass spectrometer system on the protonated molecular ion of the N-blocked tryptic peptide (MH' = 1036). In this experiment, a VGZAB 4F mass spectrometer equipped with an Ion Tech fast atom bombardment source was run in an analogous manner to the LSIMS experiments [17].only the MH' specie was allowed to pass through the first mass spectrometer, where it was collisionally activated with He at a pressure of0.1 torr. The resulting fragment ions were passed through to the second mass spectrometer and mass separated to produce a daughter ion (MS/MS) mass spectrum
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