A high-throughput capillary isoelectric focusing immunoassay for fingerprinting
protein sialylation
Lam Raga Anggara Markely1, Lila Cheung1, Young Jun Choi1, Iva Turyan2, Ruth Frenkel2, Zoran Sosic2,
James Lambropoulos3, Lia Tescione3, Melissa Berman4, Thomas Ryll1,3, Scott Estes1, and Shashi
Prajapati1,5
Supplementary Information
Supplementary Figure 1. Image of fluorescently labeled pI standards in 12 capillaries run in a single
cycle. Red arrows indicate the pI values of the standards. The pI standards were separated consistently
among different capillaries. The pI standards were used to normalize the pI values of the protein of
interest, P1, among different capillaries.
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Supplementary Figure 2. The high-throughput capillary isoelectric focusing immunoassay is sensitive
and has limit of quantitation of 12.5 µg/ml. Purified P1 standards with concentrations of 6.25, 12.5, 25,
and 50 µg/ml were analyzed by the high-throughput assay. The peak area of Peaks 1, 2, and 3 of each
standard correlated with the signal to noise ratio (A). Signal to noise ratio of 10 was used to determine the
limit of quantitation, and 12.5 µg/ml was required to obtain signal to noise ratio > 10 (B). Representative
electropherograms of 6.25 (C), 12.5 (D), and 50 µg/ml (E) purified P1 standard demonstrate that the
signal to noise ratio increased as the concentration increased from 6.25 to 50 µg/ml.
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Supplementary Figure 3. The high-throughput capillary isoelectric focusing immunoassay has different
linearity ranges for Peaks 1, 2, and 3. Peak 1 is linear from 30 to at least 50 µg/ml, while Peaks 2 and 3
are linear from 0 to at least 50 µg/ml. This difference in linearity range of Peaks 1, 2, and 3 may be
because peak area of Peak 1 is lower than that of Peaks 2 and 3. Therefore, samples were pre-diluted to a
concentration range of 30 – 50 µg/ml prior to analysis. Error bars correspond to SD (n = 6). Signal to
noise ratios of all peaks are > 10.
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Supplementary Figure 4. The high-throughput capillary isoelectric focusing immunoassay is precise.
The %Peaks 1, 2, and 3 of 31 cell culture samples had SD < 3.5%. Signal to noise ratios of all peaks
are > 10.
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Supplementary Figure 5. The high-throughput capillary isoelectric focusing immunoassay does not have
significant well-to-well and cycle-to-cycle variability. 72 replicates of the same P1 cell culture sample
were analyzed in 6 cycles, each of which consisted of 12 wells. In each of the 6 cycles, the %Peaks 1, 2,
and 3 of the 12 well replicates had SD < 3%, indicating that there was no significant well-to-well
variability. Similarly, in each of the 12 wells, the %Peaks 1, 2, and 3 of the 6 cycle replicates had
SD < 3%, indicating that there was no cycle-to-cycle variability.
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Supplementary Figure 6. The high-throughput capillary isoelectric focusing immunoassay does not have
significant day-to-day variability. The same purified P1 standard was analyzed in 10 different days, and
the SD of %Peaks 1, 2, and 3 was < 3%, demonstrating that the assay did not have significant day-to-day
variability. Error bars correspond to SD (n ≥ 3).
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