SDS-MW calibration and molecular weight estimation using
the SCIEX PA 800 Plus Empower Driver version 1.3
Firdous Farooqui1, Peter Holper1, John D. Walsh2, Handy Yowanto1
1 SCIEX, Brea, CA
2 Waters Corporation, Milford, MAIntroduction
CE-SDS, a specific CGE technique, involves the separation ofprotein-SDS complexes using a replaceable gel matrix. The gel isformulated to provide a sieving range of approximately 10 kDa to225 kDa. Within this size range, the logarithm of protein molecularweight is linear with its migration time. Therefore, the molecularweight of an unknown protein can be estimated from a standard
curve of known protein sizes1. Currently, Empower does not havepre-configured standard curve and MW estimation capability. This
technical note demonstrates the molecular weight estimation ofunknown protein peaks using Empower software CDS from data
generated with the SCIEX PA 800 Plus Empower™ Driver v1.3.

Key features
Baseline resolution of SDS-MW Size Standard peaks

Internal software calibration of SDS-MW Size Standardpeaks

Seamless integration of the PA 800 Plus System into anexisting centralized chromatography data system (CDS)Validated CE-SDS method on an automated instrument forrobust and reliable analysis

Comparable results between Empower Driver v1.3 calibration
curve (R2 = 0.999120) and 32 Karat software (R2 =0.998241).

Methods
Instrument
All experiments were performed on a PA 800 Plus System(SCIEX, Brea, CA) equipped with a PDA detector. Data wascollected using the PA 800 Plus Empower Driver v1.3. A 50 µmi.d., 30.2 cm long bare-fused silica capillary (SCIEX, P/N 338451)
was installed on the PA 800 Plus System (20 cm effective length).
The capillary temperature was maintained at 25°C in all
separations. Voltage was applied using reverse polarity.
The following methods were used with the SCIEX PA 800 Plus
Empower Driver v1.3:
 SDS MW Conditioning: conditions the capillary.

Figure 1: The PA 800 Plus Pharmaceutical Analysis System

 SDS MW Separation: performs the SDS-MW separation.
 SDS MW Shutdown: cleans the capillary at the end of thesequence for storage and turns off the UV lamp.
Chemicals and materials
SDS-MW Size Standard
The SDS-MW Size Standard contains 10 kDa, 20 kDa, 35 kDa, 50kDa, 100 kDa, 150 kDa, and 225 kDa standards and is used toestablish a calibration curve in order to estimate the molecularweight of a sample peak.
Internal Standard
A 10 kDa protein internal standard is used as a mobility marker.
The relative migration times of all protein samples are calculated
against the migration time of the 10 kDa standard, allowing for
more accurate size estimation.
Reagents and buffers
SDS-MW Gel Buffer, SDS-MW Sample Buffer, 10 kDa protein
internal standard, SDS-MW Size Standard, acidic and basic wash
solutions were all manufactured by SCIEX (Carlsbad, CA) as partof the SDS-MW Analysis Kit (P/N 390953). The IgG control
standard was manufactured by SCIEX (P/N 391743).
2-mercaptoethanol was obtained from Sigma (St. Louis, MO).
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Sample preparation

Results and discussion

Prepare the SDS-MW Size Standard

All proteins were completely separated within 30 minutes using
our recommended method. Figure 2 shows a typical separation ofthe SDS-MW Size Standard.

1.

Allow the SDS-MW Size Standard to thaw at room
temperature for 15 minutes.

2.

Mix the SDS-MW Size Standard thoroughly and centrifuge
briefly in a standard microcentrifuge.

3.

Pipette 10 μL of the SDS-MW Size Standard into a 0.5 mL
microcentrifuge tube.

4.

Add 85 μL of the SDS-MW sample buffer to themicrocentrifuge tube.

5.

Add 2 μL of the 10 kDa internal standard to themicrocentrifuge tube.

6.

Inside a fume hood, add 5 μL of 2-mercaptoethanol to themicrocentrifuge tube.

7.

Seal the vial cap with Parafilm, mix thoroughly, and heat in awater bath at 100°C for 3 minutes.

8.

Put the tube in a room-temperature water bath to cool for fiveminutes. The sample will remain stable for approximately 24hours.

9.

Transfer 100 μL of the prepared sample to a micro vial, put
the micro vial into a universal vial, and cap the universal vial.

Prepare the IgG control standard (reduced)
1.

The IgG control standard (~ 1 mL) was aliquoted into ~10vials of 95 µL each and stored at -20°C to avoid multiple
freeze-thaw cycles.

2.

Thaw one of the 95 μL aliquots of the IgG control standard atroom temperature.

3.

Add 2 μL of the 10 kDa internal standard to the IgG tube.

4.

Inside a fume hood, add 5 μL of 2-mercaptoethanol to the IgG
tube.

5.

Cap the tube and mix thoroughly.

6.

Using a centrifuge, spin the tube for 1 minute at 300 × g.

7.

Seal the vial cap with Parafilm and heat the vial at 70°C for
10 minutes.

8.

Allow the vial to cool for at least 3 minutes.

9.

Transfer 100 μL of the prepared sample to a PCR vial, put thePCR vial in a universal vial and cap the universal vial.

Figure 2: Separation of the SDS-MW Size Standard

Establishing the standard curve
A 10 kDa internal standard is used as a mobility marker and all
protein mobility is calculated relative to this internal standard. See
Figure 3 for a typical calibration curve obtained by plotting the Log
Molecular Weight versus Volume (Migration Time) of each protein
peak in the SDS-MW Size Standard. The molecular weight of an
unknown protein peak can be estimated using this calibration
curve. The calculated molecular weights are displayed in theelectropherogram by displaying the MW (Daltons) column in Table
properties. It is recommended that this curve be re-calibrated
every 24 cycles. This is done by running the SDS-MW Size
Standard and updating the calibration curve values for eachstandard to reflect the new run, as shown on Figure 3.
Figure 3 shows the SDS-MW analysis result of the SDS-MW Size
Standard, demonstrating the analysis method separates theanalytes by their molecular weights. The calibration curve
correlation coefficient (R2) generated using this analysis methodin the SCIEX PA 800 Plus Empower Driver v1.3 was > 0.99.

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R2 = 0.999120
R2 = 0.998241

Figure 4: Linear curve of log of MW vs. Migration Time from Table 2
analyzed using 32 Karat™ software.

Figure 3: Calibration curve generated by the Empower software.

The calculated molecular weight of the SDS-MW Size Standard isshown in Table 1.
Table 4: Calculated molecular weights of the SDS-MW Size

Standard using Empower software

A comparison of the R2 values generated by 32 Karat™ softwareand Empower software shows both calibration curves areidentical. Thus, the calibration curve generated using datacollected using the SCIEX PA 800 Plus Empower Driver v1.3 canbe successfully used to estimate the molecular weight of an
unknown protein peak.
Generating estimated protein molecular weights from thestandard curve

Estimated MW (Daltons) Calculated MW (Daltons)
10,00010,49820,00019,23735,00032,95950,00050,384

100,000

105,570

150,000

154,630

225,000

215,772

The Empower software data file of the SDS-MW Standard used togenerate the standard curve in Figure 3 was converted andimported into 32 Karat™ software. The file was analyzed in 32
Karat™ software to generate a calibration curve (Figure 4).

Note: High levels of glycosylation and other attributes can affectpeak migration when analyzed by CE-SDS. The molecular weight
determination is an estimation and might not be representative ofthe theoretical molecular weight of each analyte analyzed.
A glycosylated IgG standard was analyzed under reducing
conditions using the same separation conditions described above.
The molecular weight of each resulting peak was estimated using
the calibration curve generated with the SDS-MW Size Standard(Figure 5).
The following table and the annotation on each peak show theestimated molecular weight of the 10 kDa Internal Standard andeach peak of the reduced IgG standard.

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Figure 5: Molecular weight estimation of reduced IgG standardConclusions
This technical note demonstrates that data collected using theSCIEX PA 800 Plus Empower Driver v1.3 can be used to generate
a molecular weight calibration curve by plotting Log Molecular
Weight versus Elution volume (migration time) of each protein
peak in the SDS-MW Size Standard. The molecular weight of an
unknown protein can be estimated by using this calibration curve.

References
1.

SDS-MW Analysis Kit Application Guide (RUO-IDV-05-6934B)

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