Preparing Protein Complexes for Mass Spectrometry

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Protocol for Mass Spectrometry analysis of Protein Complexes:

While preparing the samples the utmost care should be taken to eliminate keratin and other contaminants from the sample. Gloves should be worn and the highest purity buffers and water should be used to prepare reagents. In essence protein samples should be treated as if you are working with RNA. Keratin is the most common contaminant and can detract from the results obtained.

Protein complex pull downs that require detergent should be washed with a buffer in the last step without detergent. For example, buffers containing 0.1% NP40 can definitely be a problem in the final elution. Initial washes can use detergent, but then include a final wash with no detergent and elute with no detergent. This only causes a problem if there are solubility problems with your complex. If you NEED detergent in the final step, you can try to titrate down the amount. After elution TCA or acetone precipitation and acetone wash of the pellet can help remove residual detergent. The acetone washes are excellent for removing small amounts of non-ionic detergents. Alternatively, you can also try to use several different mass spec friendly detergents such as Invitrosol (https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=12421&CMP=LEC-GCMSSEARCH&HQS=invitrosol).

It is also helpful to run 10-20% of the final elution step on a silver stained gel to estimate the amount of protein present. Send the dry pellet.