Protein Extraction Mechanical grinding Detergents Other buffers Sonication httpwwwpiercenetcombrowsecfmfldIDFA97D803695348E4A7BD6947D35FE83B Considerations for mass spectrometry Salts and buffers in high concentrations can cause ion suppression and adduct formation in electrospr ID: 316190
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Slide1
Considerations for Sample PreparationSlide2
Protein Extraction
Mechanical grinding
Detergents
Other buffersSonicationSlide3
http://www.piercenet.com/browse.cfm?fldID=FA97D803-6953-48E4-A7BD-6947D35FE83BSlide4
Considerations for mass spectrometry
Salts and buffers in high concentrations can cause ion suppression and adduct formation in electrospray mass spectrometry
Detergents can interfere with reversed phase
separations
ProteasesSlide5
Inhibitors
Most cells have endogenous proteases that can indiscriminately cleave proteins once cellular structure is disrupted
The same applies to many PTMs (phosphatases,
deacetylases
,
etc
)Slide6
SaltsSlide7
Medicago with detergentSlide8
MS 2 of detergentSlide9
Lysis buffer
Compatible
lysis
buffer8 M Urea, 50 mM Tris pH 8.5, 5
mM
CaCl
2
, 50-100
mM
NaCl
, plus inhibitorsSlide10
Detergents
Mass Spec compatible:
RapiGest
(Waters)ProteaseMax
(
Promega
)
For other detergents, you need a clean up step prior to LC-MS
FASP
Precipitation (acetone, chloroform/methanol)Slide11Slide12
Desalting
C18 reversed phase materialSlide13Slide14Slide15Slide16Slide17