Friday, January 8, 2016
Should you take a second look at your PepMap gradient?
Check this out! This is the same amount of the Thermo HeLa peptide mix ran on the same instrument but using two different gradients. (Click to expand)
Gradient 2 picked up 2,000 more peptides that translated into over 400 (don't check my arithmetic please, it is really early here) new proteins! Sure, they're probably single peptide hits, but I bet if you looked at them you'd see they're from low abundance (translation: cooler!) ones.
How'd the awesome scientists running this optimization get this boost? By changing their gradient from this:
2-35% B in 90 minutes
2-20% B in 75 minutes 20-32%B in 15 minutes.
Holy cow! That's a two thousand peptide gain? Totally for free! It looks like C-18 PepMap material (like that used in the EasySpray columns) might be a little more hydrophilic (? or something?) than other C-18 materials like C-18 Magic or older materials. Or two stage gradients are just awesome. I don't know, but if you've got the time maybe you should investigate a more shallow gradient.
P.S. Useful information:
Channel A -- 0.1%FA
Channel B -- 100% ACN 0.1%FA
Flow rate 300nL/min
200ng HeLa digest on column
QE Plus running a standard "Top10" method
Big ShoutOut to: Tara, Josh, and Lani cause they did the work. I just stole from their slides!