Waters MassPREP Enolase Digest with Phosphopeptides Mix - Manual (Page 3)

[ Care and Use ManUal ]
MassPREP Enolase Digest with Phosphopeptides Mix 3
ii. For separations on columns less than 200 µm in diameter,
the recommended injection volume is 1 µL of a solution
200 fmol/µL or more.
IV. example speCt ra
The following MALDI TOF MS spectra of MassPREP Enolase Digest
with Phosphopeptides Mix (10 pmol/sample well) mixed with
MassPREP MALDI Matrix - DHB (20 mg/mL) was processed by
baseline subtraction. Data in positive reflectron mode was obtained
on a Waters MALDI micro MX (sum of 200 laser shots). Where
indicated, some samples were analyzed using 1% phosphoric acid
(PA) in the solvent mixture used to solubilize the DHB matrix. In
addition, a number of commercially available phosphopeptide
enrichment strategies can be used to selectively improve the signal
intensity of the phosphopeptides.
Figure 1: Enolase Digest with Phosphopeptides, 600 to 2600 Da
Figure 2: Closer view of T43 1P and T34 2P
Figure 3: Closer view of T18 1P and T19 1P
The following LC-ESI-MS (TIC) and LC-UV (at 210 nm) spectra
were obtained using a Waters Alliance
® 2795 coupled to a Waters
ZQ 4000 mass spectrometer. A 20 pmol/μL solution in 0.02%
trifluoroacetic acid in water was prepared and 30 μL was injected on
a Waters BioSuite™ C
18
3 μm, PA – A, 2.1 x 150 mm column. Data
from the Waters MassPREP Phosphopeptide Standard – Enolase is
included in the following figures in order to more clearly identify the
phosphopeptide LC peaks.
Figure 4: LCMS graph
Figure 5: LCUV graph
m/z
600 800 1000 1200 1400 1600 1800 2000 2200 2400
%
0
100
%
0
100 807.3
800.2
756.3
808.3
1579.2 1288.9 1159.8
814.3
1057.7
1161.8
1368.9
1756.4 1581.2 1841.4
2442.9 2329.8
756.3
726.1
723.2
807.3
808.3
1413.1
1159.7
814.3
1057.7 1288.9 1841.4
1579.2
1778.4
2442.9
1896.5
DHB with 1% PA
DHB
m/z
1350 1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480
%
0
100
%
0
100 1368.9
1357.9
1417.0 1413.1
1369.9
1370.9
1373.0
1418.0
1449.0
1419.0
1420.0
1451.0
1413.1
1368.9
1357.9
1390.9
1371.0
1398.1 1406.9
1414.1
1417.0
1439.0
1435.1 1419.0 1449.0 1471.0
1461.1
T43 1P
T43 2P
T43 1P
T43 2P
DHB with 1%
PA
DH
B
m/z
810 815 820 825 830 835 840 845 850 855 860 865 870
%
0
100
%
0
100 814.3 809.3
810.3
815.4
816.4
863.3
814.3
809.3
810.2
836.3 829.3
822.2 815.4
816.3
823.2
827.3
835.2
830.3
832.3
863.3
837.3
857.3 842.4 851.3 844.2 859.4
864.3
868.2
870.3
T18 1P
T19 1P
T19 1P T18 1P
DHB with 1%
PA
DHB
Time
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
LCMS
T18 1P
T43 2P
T19 1P
T43 1P
T18 1P T43 2P T43 1P w/enola se peptides T19 1P w/enola se
peptides
MassPREP Phosphopeptide Standard - Enolase
MassPREP Enolase Digest with Phosphopeptides Mix
MassPREP Phosphopeptide Standard - Enolase
MassPREP Enolase Digest with Phosphopeptides Mix
Time
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
LCUV
T18 1P
T43 2P
T19 1P
T43 1P
T18 1P
T43 2P
T43 1P w/enola se peptides
T19 1P w/enola se
peptides