Analysis of rates of multiple enzymes in cell lysates by electrospray ionization mass spectrometry [8]

Journal of the American Chemical Society

Volumn 121, Issue 5, 1999, Pages 1102-1103

  Gerber, Scott A ^a   Scott, C Ronald ^b   Turecek, Frantisek ^a   Gelb, Michael H ^a,b  

^a UNIVERSITY OF WASHINGTON   (United States)

^b UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ALPHA N ACETYLGLUCOSAMINIDASE; BETA GALACTOSIDASE; LYSOSOME ENZYME;

CELL LYSATE; ENZYME ACTIVITY; ENZYME ANALYSIS; ENZYME DEFICIENCY; ENZYME SUBSTRATE; INBORN ERROR OF METABOLISM; LETTER; LYSOSOME STORAGE DISEASE; MASS SPECTROMETRY;

EID: 0033540716     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja982878k     Document Type: Letter

References (18)

1. Scriver, C. R.; Beaudet, A. L.; Sly, W. S.; Valle, D. The Metabolic and Molecular Bases of Inherited Disease; McGraw-Hill: New York, 1995.
2. Scriver, C. R.; Kaufman, S.; Eisensmith, R. C.; Woo, S. L. C. In The Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 1015-1076.
3. Segal, S.; Berry, G. T. In The Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill; New York, 1995; pp 967-1000.
4. Chen, Y.-T.; Burchell, A. In The Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 935-966.
5. For example, see: Morris, A. A. M.; Turnbull, D. M. Curr. Opin. Neurol. 1994, 7, 535-541.
6. Cole, R. B. Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Practice; Wiley: New York, 1997.
7. Okada, S.; O'Brien, J. S. Science 1968, 160, 10002.
8. Neufeld, E.; Muenzer, J. The mucopolysaccharidoses. In The Metabolic and Molecular Bases of Inherited Disease, Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 2465-2494.
9. Bayer, E.; Wilchek, M. Methods Enzymol. 1990, 184, 49-51.
10. Wilbur, D. S.; Hamlin, D. K.; Pathare, P. M.; Weerawarna, S. A. Bioconjugate Chem. 1997, 8, 572-584.
11. 3CN and catalytic NaOH.
12. 1H NMR and ESI-MS.
13. Romanowska, A.; Meunier, S. J.; Tropper, F. D.; Laferriere, C. A.; Roy, R. Methods Enzymol. 1994, 242, 90-101.
14. Cell protein (75 μg) in 15 μL of water was added to 15 μL buffer (0.1 M Na citrate, pH 4.25) containing 2 (0.3 mM) and 1 (0.2 mM, added 5 h after addition of cell protein). After incubation for 5.5 h at 37°C, the reaction was quenched by addition of 200 μL of 0.2 M glycine carbonate buffer, pH 10.3, and 5 and 6 (1 nmol each) were added. After centrifugation to remove cell debris, the supernatant was loaded onto a bed of streptavidin-agarose (7 nmol biotin binding capacity, Pierce) in a small filtration device (micro BioSpin, Bio-Rad). After 5 min, filtration was effected by centrifugation, and the gel bed was washed with 0.1% Triton X-100 (∼1 min incubation, then spin) and then six times with purified water (Milli-Q, Millipore). Elution was carried out in 25 μL of 50% methanol containing 56 nmol of free biotin (1-10 h incubation, then spin). Filtrate was diluted 4-fold with 50% methanol/ water, and 1 μL was analyzed by ESI-MS.
15. 2-OH) showed quantitative streptavidin capture efficiency from a cell homogenate.
16. Radioactive model conjugate (see footnote 15) was released in ∼85% yield after incubation with excess biotin for 90 min.
17. ESI-MS was carried out on a Finnigan LCQ ion trap instrument. Data were collected in full scan mode from m/z 625 to 875 by direct infusion at 1.5 μL/min. Specific activities were obtained from the ratio of product to internal standard ion peak areas (averaged over 30 scans).
18. Time course studies confirmed that the initial reaction velocities were being measured.