Help - Protein not binding to C4, C18

[HAZMAT]

"am experiencing lack of protein binding to C4 and C18 trap columns. I am optimizing the method using BSA at 2pmol/ul in 0.5% ACN/0.1% FA. 10ul (= 20pmol = 1.34ug on trap) are loaded on either C4 or C18 at 80ul/min connected to a ESI-QTOF. The protein elutes after 0.45 min (= dead volume of the LC system). The loading solvent is 10% ACN/0.1% FA."


"The method for binding proteins to RP material is well established, however I am not sure what is leading to the lack of binding? The percentage of organic solvent in the sample is low (+/- 1-5%) and the pH should be acidic due to the presence of 0.1% FA. Under these conditions proteins should bind to C4 and C18 material. Perhaps using some TFA instead of FA? "

[Uwe Neue]

Why do you use 10% MeCN? What is the pore size of the packings?

[HW Mueller]

Also, there is hydrophobic interaction chrom. (HIC) which shows that some proteins need to be forced to adsorb to reverse phase surfaces.

[Kostas Petritis]

I haven't see many proteins that are eluted in only 10% ACN at reverse phase conditions but I would start even lower just in case... and as Uwe is implying your protein might be excluded from the column if your pore size is too small...

[danko]

“Excludedâ€

[HW Mueller]

One can even have ion exclusion, the protein coming out ahead of tm (to). I have seen this with Mab on reverse phase during those trials where I unsuccessfully tried to do the Mab isocraticaly on C-18.

[HAZMAT]

Thank you all for your feedback.

It looks like we're using a material with a 100 Angstrom pore size. BSA is about 70 kDa, do I need a 300 Angstrom pore size to retain this protein?

I'll suggest starting with a 5% ACN mobile phase to see if it has any effect.

[Uwe Neue]

You will avoid a lot of problems in the future if you use a 300 A material instead of the 100 A material.

[HAZMAT]

We will pack the 300A material and give that a try. Also, we will experiment with a 5% ACN mobile phase. I'll followup with results ASAP.

[Bryan Evans]

Below is bovine serum albumin injected on to Intrada WP-RP (30nm pore size):
http://www.imtaktusa.com/site_media/fil ... TI289E.pdf

[HAZMAT]

"I moved away from testing with BSA to my real samples. These proteins are more hydrophobic and hence I'm getting better results. I am using H2O/0.1% FA to load the samples and 90% ACN/5% FA to elute. This is giving me reasonably good results however with 5% FA my MS spray is not great (0.1% -1% FA results in much better spray and hence ionization)."