Followed instructions for enriching 1 mg HeLa + pStd. After elution dried down in speedvac and was somewhat "jelly", and brought up in 40 ul of 95/5 0.1% formic and it came up just fine with some pipet mixing. Approximately 60 ul total transfered to autosampler. 

Injected 1 ul for a 120 min DDA run, and based on how 200 ng HeLa looks, expect was 100 ng/ul.


(unless specied in file name 100 ng, which is an estimate, all were 4 ul which looks to be about 400 ng on column)



Running ultimate 3000 over a trap then column.
PepMap 100 C18 trap column (75 µm id x 2 cm length; Thermo Fisher Scientific)
Acclaim PepMap RSLC 2 µm C18 column (75µm id x 25 cm length; Thermo Fisher Scientific)


loaded onto a PepMap 100 C18 trap column (75 µm id x 2 cm length; Thermo Fisher Scientific) at 3 µL/min for 10 min with 2 % acetonitrile and 0.05 % trifluoroacetic acid followed by separation on an Acclaim PepMap RSLC 2 µm C18 column (75µm id x 25 cm length; Thermo Fisher Scientific) at 40 °C.  


Gradient (all gradients were the same)
Loading is 10 min at 3 ul/min
120 min gradient of 5 % to 27.5 % mobile phase B (80 % acetonitrile, 0.08 % formic acid) over 105 min followed by a ramp to 40 % mobile phase B over 15 min at a flow rate of 300 nL/min. 

time	%B
0	5
10	5
115	27.5
130	40
140	95
150	95
150.1	5
160	5


data acquired from 10 to 150 (so yeah, 140-150 is some junk)


MS/MS Settings

Will add more specifics on DDA only run, or the DDA + targeted list HERE (later). 

The GPF samples were all run according to notes. 1 - 300-400.... 8 - 1100-1200. 


The DIA method is the staggered 8 m/z windows running 60k/15k res OTOT.


The surequant run was on 7/9 and used the method from Tony, but with my gradient and using 4 ul injection. The instrument seemed to be running well enough (HeLa was still at ~4100) but I really can't eval the method based on the run.