Hi Jaekwan Kim,

my first question is on howyou performed the nanodrop analysis of the peptide concentration? Have you set the baseline (Blank) correctly by using the respective buffers for the different solutions (sample loading buffer, washing buffer, elution buffer). If you have done the blank measurement with dest Water for all solutions your results might be not correct.

However, there are some point in the stage tip procedure that can be the reason of bad recovery.

The first is if you put the C18 disks to loose into the tip there might be a leak where the buffer can flow through without retention of the peptides at the C18 matrix. The sam will happen if the disk stacks of the produced stage tip has wrinkanges at the side (by usage of a two large diameter of the blunt ended hamilton syringe needle for punching out the disks and push into the tip / 16gauge size in the original protocol Rappsilber et al. 2007). I would allways check freshly prepared stage tips for a leakage with a short centrifugation step before possibly wasting important samples.

Another point which is rarely addressed in protocols is the activation state of the C18 matrix in the stage tip which is active for proper peptide bilding/retention as long as the material remains wet. I.e, if you activate your C18 matrix with 100% methanol but leave the tips for a longer time before proceeding with the next step, so that the matrix dries and the C18 become inactive and your peptides will flow through. The drying of the C18 can also happen if your centrifugation steps between activation and sample loading are too long or the centrifugation speed is to fast so that every liquid is drained away from the C18. The setup of the centrifugation time and speed is depending on how tight the C18 disk stacks are packed (which could be very variant). The tighter the stacks in the stage tip are packed the longer the centrifugation or the higher the speed will be required. For this reason I would recommend (and also because of the drying issue mentioned above) to evaluate time and speed for the individual type of tightness of the stage tips.

Good luck,

Murat

Murat Eravci

Hi Murat Eravci.

I am really appreciated for your advise.

what you first mentioned about Blank is not the problem as after collecting sample, wash, elution, vac-dry was performed and all samples were recontitted with same solution.

and centrifugation step between activation and sample loading is short, so it is not problem.

I think i put the C18 disks to loose into the tip....

because the manual said "gently pack the material" (see the below manual)

https://www.upstate.edu/proteomics/pdf/stage_tip_c18protocol_w_photos.pdf

you mean pack the C18 disks to tight by pressing the disk strongly?

I will try it

Thank you again.

Sincerely

Hi Jaekwon Kim,

The disks should be placed firmly in the tip end. If you do too gentle there might be leakages or the disks will float after pipetting the solutions (happens to a student during one of my practicle MS courses). If you push or press the disks to tight or to strong the stage tip can be clogged very fast leading to very long centrifugation times. Maybe with some practice you will find out the adequate force to pack the c18 disks into the tip end. Btw, how many disks are you usually pack into one stage tip? I usually do a stage tip with 3 disks (all placed into the tip end one after the other never all three together what also improves the production of leakage free stage tips).

Best,

Murat

Murat Eravci Hi Murat Eravci

Thank you for your kind reply.

I usually do a stage tip with 3 disks

My disk product number is this (CDS Empore 2215: C18-HD bonded silica, 12um particle size. SPE disk, 47mm #98-0604-0217-3)

As i tried pressing the disks with varing degrees of intensity, I think it is disk problem.

So, I will open new disk package and try it one more time.

If you have any other idea, please let me know.

Thank you again.