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MALDI interfacing and miniaturization

MALDI interfacing and miniaturization. Proteomics 2002, 2, 360-372. 생명과학부 박사과정 구용의. Contents. Introduction Fraction collection coupling Continous sample desorption Mass spectrometric imaging Microfabricated systems Conclusion. Signal suppression.

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MALDI interfacing and miniaturization

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  1. MALDI interfacing and miniaturization Proteomics 2002, 2, 360-372 생명과학부 박사과정 구용의

  2. Contents • Introduction • Fraction collection coupling • Continous sample desorption • Mass spectrometric imaging • Microfabricated systems • Conclusion

  3. Signal suppression • MS signals of individual species do not correspond to their respective conc. • Due to 1) charge competition 2) the degree of analyte incorporation into matrix crystals • Increase with sample complexity  require sample separation

  4. Signal suppression in MALDI-TOF analysis Each 1M 50M+1M

  5. MALDI vs ESI

  6. Fraction collection coupling for MALDI • Coupling of separation with MS  most powerful analytical system • Separation of sample minimize signal suppression • MALDI is performed in deep vacuum  off line by collecting fractions

  7. Advantage of fraction collection • Analysis by other procedure • Protein sequence coverage and detection of large protein fragments • Various column • Additional clean-up or protein digestion

  8. 2-Dimensional separation • 2-D PAGE • 2-D chromatography(Fig. 2.) • Solution IEF+SDS electrophoresis or reversed phase separation • Solution IEF + MALDI-TOF MS(Fig. 3.)

  9. 2-D separation by SEC + RPLC

  10. pI-MS 2-D image

  11. Continous sample analysis • Frit and aerosol desorption • Surface deposition • Mechanical systems • Electrospray deposition

  12. Frit and aerosol desorption(With probe) • A stream of sample in a liquid matrix (3-nitrobenzyl) is delivered through the probe • The analyte is desorbed directly from the frit on the probe tip • Excess liquid is removed with paper at the end of the probe

  13. Frit and aerosol desorption(With aerosol) • MALDI is perfomed directly on the mist of rapidly dried droplets (nebulizer). • Simple, but inefficient and poor sensitive, low resolution

  14. Surface deposition • In-line approach eliminating the disadvantages of both discrete fraction collection and on line coupling • Solidified samples can be analyzed immediately or can be archived for long periods of time for additional tests.

  15. Mechanical systems • Continous deposition on a wetted cellulose target coated with matrix 2) The roatating ball design(ROBIN interface) 3) The vacuum deposition interface with tape cartridge(Fig. 4, 5, 6) - high resolution - homogeneous uniform sample trace

  16. Electrospray deposition • For deposition of a homogeneous sample layer for laser desorption • More homogeneous, better quantatitive, less various • Disadvantage - the wide spreading of sample over the MALDI target  electrofilament mode

  17. Mass spectrometric imaging • MS images of samples in one or more m/z (sample+MALDI)  2-D maps(Fig. 7.) • as a diagnostic tool for testing of sample preparation protocols, sample morphology, and spectral quality of MALDI samples • Virtual 2-D gel analysis(Fig. 8.) - run on IPG strip first - scanned with MALDI beam

  18. Microfabricated systems • The enabling technologies for the current explosive growth of the biological sciences • Benefits - space saving and cost reduction - faster analysis - lower sample and reagent consumption - more sensitive and repeatable

  19. Automatic microanalysis system

  20. Silicon chips with immobolized target DNA • Amplified DNA fragments covalently attached to chip well • Primer annealing, extension, termination • Detected in situ by MALDI-TOF  accurate, high-throughput, low cost identification of genetic variation

  21. Protein chips • Rapid protein analysis and expression profiling  Revolutuional proteomics research • For expression monitoring or diagnostic purpose • The reading of the array  optical imager or MS detection

  22. Conclusion • High throughput, high resolution analysis of minute sample  by new interfacing technique  by new ionization technique

  23. Urinary proteome(Proteomics 2001, 1, 93-107,108-117) • Easily accessible sample in human metabolism, toxicology and disease • HSA is predominant in urine • DEX(dynamic exclusion) - direct LC-MS/MS - the exclusion of an ion from the data-dependent ion selection for a given period of time

  24. DEX • process which places the set mass of previously selected precursor ions into a time-dependent reject mass list to reduce redundant fragmentation • The pursuit of ions present at less than the base peak intensity

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