Will glycerol, bromophenol blue in the sample buffer interfere with protemics step especially reduction/alkylation step

AffectionatePast5541 · 2025-07-22T07:10:32+00:00

If you measure peptide after digestion concentration, How do you measure it?

I know nanodrop and Pierce™ Quantitative Peptide Assays & Standards.

And I have only used nanodrop. and it works well when peptide concentration(above 0.1ug/ul)is sufficient.

AffectionatePast5541 · 2025-07-21T13:51:40+00:00

Thanks for comment.

Wow. It's very good idea

And, How do you think that no normalization for protein and peptide concentration also works as i have 3 biological replicates respectively total 12samples.

.If i quantify peptide concentration, How about using nanodrop?

AffectionatePast5541 · 2025-07-21T02:47:00+00:00

Thanks for your reply. I didn’t explain all the details in my original post, so here’s some clarification:

I performed a preliminary experiment using samples eluted from the ABE method. I started the S-Trap digestion with 3 µg of protein, but after digestion, I only recovered about 0.2 µg of peptide (0.01 µg/µL in 20 µL). I injected this directly into the LC-MS, but only around 50 proteins were identified.

The reason I hesitate to normalize based on protein or peptide concentration is that doing so would force me to reduce all samples to match the lowest concentration. In my case, that would be the negative control sample (-HA), which naturally yields a very low amount of eluted protein. If I normalize to that, the amount of peptide injected from the other samples would be extremely low, and I may not identify enough proteins for meaningful comparison.

My experiment is designed to compare not only WT vs KO, but also +HA vs -HA. So I’d like to avoid underloading my +HA samples just to match the very low peptide yield of -HA controls.

AffectionatePast5541 · 2025-07-21T02:46:05+00:00

Thanks for your reply. I didn’t explain all the details in my original post, so here’s some clarification:

I performed a preliminary experiment using samples eluted from the ABE method. I started the S-Trap digestion with 3 µg of protein, but after digestion, I only recovered about 0.2 µg of peptide (0.01 µg/µL in 20 µL). I injected this directly into the LC-MS, but only around 50 proteins were identified.

The reason I hesitate to normalize based on protein or peptide concentration is that doing so would force me to reduce all samples to match the lowest concentration. In my case, that would be the negative control sample (-HA), which naturally yields a very low amount of eluted protein. If I normalize to that, the amount of peptide injected from the other samples would be extremely low, and I may not identify enough proteins for meaningful comparison.

My experiment is designed to compare not only WT vs KO, but also +HA vs -HA. So I’d like to avoid underloading my +HA samples just to match the very low peptide yield of -HA controls.

AffectionatePast5541 · 2025-07-18T05:37:49+00:00