Application Update 188
AU70140_E 08/12S
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With the next-generation MabPac
™
SCX-10 (3 µm)
column, both separation efficiency and resolution can be
further improved (Figure 5). Separation time can be easily
reduced to <10 min by simply increasing the flow rate.
Additionally, the peptides have better retention and are
better resolved on the MabPac SCX-10 column compared
to the ProPac SCX-10 column.
For the synthetic peptide and byproducts separation, the
ProPac SCX-10 and MabPac SCX-10 (3 µm) columns
provide similar resolution (Figure 6). The latter column
performs better for resolving the main product (Peak 1)
and a nearby byproduct (Peak 2).
Conclusion
This work shows that the ProPac SCX-10 column
delivers high-resolution separations for peptide mapping
and provides an alternate or supplementary method
for reversed-phase peptide separation. The new
MabPac SCX-10 (3 µm) column delivers faster high-
resolution peptide mapping. Both columns can be used to
separate a synthetic peptide and its byproducts, providing
an alternative to reversed-phase separation.
References
1.Dionex (now part of Thermo Scientific) Application
Note 521: Automated 2D LC Coupled to ESI-MS/MS
for the Analysis of Complex Peptide Samples.
Sunnyvale, CA, 2002. [Online]
webdocs/5476-AN521_LPN1470.pdf (accessed June
18, 2012).
2.Dionex (now part of Thermo Scientific) Application
Note 126: Determination of Hemoglobin Variants by
Cation-Exchange Chromatography. Sunnyvale, CA,
2007. [Online]
webdocs/4466-AN126_released022707.pdf (accessed
June 18, 2012).
3.Dionex (now part of Thermo Scientific) Application
Update 183: Separation of Peptides from Enzymatic
Digestion of Different Acclaim Columns: A
Comparative Study. Sunnyvale, CA, 2011. [Online]
-
Peptides-EnzyDigest-AcclaimCompar-02Nov2011-
LPN2973.pdf (accessed June 18, 2012).
4. Dionex (now part of Thermo Scientific) Technical Note
74: High Peak Capacity Nano LC Peptide Separations
Using Long Packed Columns. Sunnyvale, CA, 2009.
[Online]
-
TN74-HPLC-Peptides-LongColumns-29Jan09-
LPN2111.pdf (accessed Aug 8, 2012).
Figure 6. Chromatograms of a synthetic peptide and its byproduct using 1) a
ProPac SCX-10 column (10 µm particle size) and 2) a MabPac SCX-10 column
(3 µm particle size)
200
mAU
-10
100
mAU
-10
0
5
10
15
20
25
30
Minutes
(1)
(2)
Columns:
1
. ProPac SCX-10, 10 µm (4 × 250 mm, P/N 075725)
2
. MabPac SCX-10, 3 µm (4 × 50 mm, P/N 077907)
Mobile Phase:
A: 20 mM TEAP, pH 3.9, 50% acetonitrile
B: 100 mM sodium perchlorate in A
Gradient:
1
. 0–20 min, 0–100% B, at 1.4 mL/min
2
. 0–15 min, 0–100% B; 15.5–25 min, 0% B, at 0.4 mL/min
Inj. Volume:
20 µL
Temperature:
30 °C
Detection:
UV, 214 nm
Sample:
A synthetic peptide and its byproducts
The sequence of the peptide:
(Ac-)YNIQKESTLPLVLRLRGG(–CONH
2
)
Calculated pI of the peptide: 11.4
Peak X is also shown in the blank, so it should not be considered a real peak.
X
2
1
2
1
Figure 5. Chromatograms of a myoglobin tryptic digest separated by 1) a
ProPac SCX-10 column (10 µm particle size) and 2) a MabPac SCX-10 column
(3 µm particle size).
100
mAU
-10
50
mAU
-10
0
5
10
15
20
25
30
Minutes
(1)
(2)
Columns:
1
. ProPac SCX-10, 10 µm (4 × 250 mm, P/N 075725)
2
. MabPac SCX-10, 3 µm (4 × 50 mm, P/N 077907)
Mobile Phase:
A: 20 mM TEAP, pH 2.0, 50% acetonitrile
B: 100 mM sodium perchlorate in A
Gradient:
1
. 0–20 min, 0–100% B, at 1.4 mL/min
2
. 0–15 min, 0–100% B; 15.5–25 min, 0% B, at 0.4 mL/min
Inj. Volume:
20 µL
Temperature:
30 °C
Detection:
UV, 214 nm
Sample:
Myoglobin (from equine heart) tryptic digest
Sample Preparation: Reduce, alkylate, dialyze, and digest with trypsin overnight
