1 / 32

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.

sophia-roy
Download Presentation

MALDI interfacing and miniaturization

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related