Microchannel plate for surface-induced dissociation in mass spectrometry.

A new method for surface-induced dissociation of molecular ions, applied to tandem mass spectrometry, is achieved by collisions at a grazing angle on the inside channel surfaces of a microchannel plate. This technique, termed microchannel SID, is demonstrated by using both positive and negative parent ions in the energy range of 500-2000 eV. Fragmentation spectra of the pentapeptide leucine-enkephalin (555 daltons) at 500 eV show good sequence information with a net fragmentation efficiency of 14%. High mass fragmentation is demonstrated on (CsI)23Cs+ (6113 daltons), with the resultant spectrum showing all cluster fragments from n = 0 to 23.

Revision of the blocked N terminus of rat heart fatty acid-binding protein by liquid secondary ion mass spectrometry.

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.

Evidence for isoaspartyl (deamidated) forms of mouse epidermal growth factor.

A variant form of mouse submaxillary gland epidermal growth factor (EGF) was identified by isocratic reversed-phase HPLC of EGF obtained by Bio-Gel P-10 column chromatography ("culture grade"). The variant form was essentially absent in preparations of EGF further purified by chromatography on DEAE-cellulose ("receptor-grade" EGF). The spectral properties and amino acid composition of the variant form (EGF-I) could not be distinguished from those of the intact polypeptide isolated by HPLC (alpha-EGF). Receptor-binding and mitogenic properties of EGF-I were also equivalent to those of alpha-EGF. These data suggested that EGF-I was structurally very similar to EGF. However, the very low yield (less than 4%) obtained by Edman degradation indicated that the N-terminal (Asn1) of the polypeptide was modified. Isoelectric focusing of EGF-I revealed two major immunoreactive bands: one with a pI equivalent to that of alpha-EGF (pI 4.6) and another at pI 4.1. Alkaline treatment of alpha-EGF (0.1 M NH4OH) yielded peak material by HPLC that coeluted with EGF-I; the alkaline-generated EGF-I yielded bands that also focused at pH 4.6 and 4.1. Ammonium hydroxide treatment of [des-Asn1]-EGF (beta-EGF) did not produce conversion to EGF-I. On the basis of these data, we propose that EGF-I was formed by selective deamidation of the N-terminal Asn of intact EGF. This notion is also supported by liquid secondary ion mass spectrometry, which showed that EGF-I was approximately 1.5 mass units greater than alpha-EGF. The heterogeneity observed by isoelectric focusing supports previous studies which have shown that, following deamidation of N-terminal asparagine, a beta-aspartyl shift can occur, which in the present study might yield succinimido-aspartyl1-EGF and beta-aspartyl1-EGF. Low yields observed during Edman degradation indicate that negligible amounts occur as the alpha-aspartyl1-EGF isomer.

Profile analysis of volcano field ionization mass spectra of urine from mice with sarcoma I transplanted by intraperitoneal inoculation.

A group of 20 inbred mice were inoculated intraperitoneally with sarcoma I cells. Urine specimens were collected before and after inoculation from this group as well as from a group of 20 mice for controls. Mass profiles of these groups were obtained by volcano field ion mass spectrometry and compared. Significant differences in average normalized mass peaks between the two groups after inoculations are observed for most of the peaks analysed from mass 84 to 313. There is sufficient diagnostic power in the mass profiles to permit unambiguous separation of the diseased mice from the healthy ones.

Normalization and reproducibility of mass profiles in the detection of individual differences from urine.

The mass profiles of urine samples and their reproducibility (coefficient of variation) obtained by field-ionization mass spectrometry (volcano source) depend upon the choice of normalization method. We compared six different normalization techniques in terms of improved reproducibility and sensitivity. Several simple procedures markedly improved reproducibility while preserving the high degree of sensitivity. Although these procedures gave similar and satisfactory results, others were clearly inferior and are not recommended for clinical profiling.

Characteristics of a volcano field ionization source mass spectrometer operating at 30 kV accelerating voltage.

A volcano ionization source has been coupled to an E x B velocity filter type mass spectrometer. The principal advantages of this instrument are its high efficiency, measured at 10(-5) ions per molecule for toluene (m/z 92), and long source lifetimes of over 200 h. The mass spectrometer is designed primarily to obtain mass profiles from multicomponent samples. These profiles are obtained from untreated urine and blood serum (1 microliter and 0.25 microliter samples), then measured between m/z 50 and 400, and are finally analyzed by computer.