I've been using this protocol but i'm unable to get back the maximum protein yield in Nano ESI Q TOF LC-MS/MS
The pipeline goes this way:
1. cell line lysis and homogenization
2. acetone precipitation
3. protein was diluted in 100ul of 100mM Ammonium Bicarbonate buffer
(*can we use some other buffer to dilute the protein sample? if yes, then which 1?)
4. Bradford analysis
the protein concentration obtained was 4743ug/ml
5. 100ug of protein was later digested using trypsin promega gold overnight and the reaction was stopped on the next day using 0.1% TFA followed by desalting using the following protocol.
6. and then the obtained sample in 50% ACN was rediluted using 0.1% formic acid
The ziptip used here is: Z720070 SIGMA Millipore® Ziptips C18, pack of 96 Synonym: pipet tips, pipette tips
*when do we carry out the speed vac centrifugation?
is there a need for it?
*my query is how can we determine the amount of protein loss?
*can protein concentration be determined after digestion and desalting prior to injection in lc ms ms? if so then how can i do it?
Dear Anjali,
the binding capacity of your Ziptip is about 5µg. Why do you go into this with 100µg tryptic peptide if you take care of your protein loss? In this case you will loose 95% because of the Ziptip capacity, right?
Normaly 5 to 10µg of tryptic peptides will be enough to have good results in LC-MS experiments. Depending on the length of your LC separation and the column you use you can also go up with your peptide input. If so I would recommend a solid phase extraction procedure with a higher binding capacity, i.e. C18 empore disk cartridges.
Best, Murat
Sir,
The zip code tip protocol was performed using only 10ul of the digested sample not the complete digest sample.
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