Fractions from the regions indicated in Figure 3 were collected, prepared, and enzymatically digested with trypsin (Table
1). An aliquot was removed from each sample digest and analyzed by LC-MS. Protein identifications for the chromatographic
fraction at time range of 19 to 21 min (Figure 3) are listed in Table 3. These results confirm obvious differences in proteins
between the samples as indicated in the bracketed region of the chromatogram, amplified in the inset. The cortisol-deficient
serum sample had no complement H or apolipoprotein H, while the high rheumatoid factor serum showed a relative loss in complement
H, as noted by the lower number of spectra found in comparison with the control serum.
Table 3. Protein Identifications from Fractions of Each Serum in the Time Range of 19-21 Min
A similar analysis of the chromatographic fraction from 29 to 32 min, also distinguished by differences in UV absorbancy (Figure
3), was performed on the three samples (Table 4). The bracketed region of the chromatogram shows a difference in protein levels
between the control serum and the abnormal sera. The high rheumatoid factor serum has a lower level of complement component
3 compared to the control or cortisol-deficient serum. Both cortisol-deficient and high-rheumatoid-factor serum show a decreased
level of complement component 4A, while cortisol-deficient serum is decreased in apolipoprotein A-1 protein levels. Several
of these proteins are found at reduced levels in the sera of patients with systemic lupus erythematosus, rheumatoid arthritis,
and scleroderma.2 The same proteins were identified after immunodepletion by mRP-C18 fractionation and screening comparisons of the chromatographic
UV traces of suspect and control sera prior to confirmation by LC-MS.
Table 4. Protein Identifications from Fractions of Each Serum in the Time Range of 29-32 Min
The mRP-C18 column optimizes fractionation of human serum proteins after depletion of high-abundance proteins that interfere
with the detection of low-abundance protein components of the plasma proteome. The ability to integrate immunodepletion and
fractionation permits high recovery, excellent resolution, and reproducibility in reducing sample complexity for biomarker
research while accelerating analytical throughput. The greater than 98% protein recovery from the immunodepleted serum makes
it possible to validate the characterization and comparison of serum samples through differential expression of biomarkers.
In addition, reproducible protein fractionation with high resolution provides the means for rapid screening of samples by
UV absorbancy, reducing sample complexity, and permitting a more effective targeted selection of samples for subsequent LC-MS
analysis. Application of this technique may extend beyond sera fractionation and could prove useful in other biologic sample
William Barrett is a product manager at Agilent Technologies, 2850 Centerville Road, Wilmington, DE 19808-1610, 302.633.8120, fax 302.993.5949,
James Martosella is an R&D scientist at Agilent, James_martosella@agilent.com
Nina Zolotarjova is an R&D scientist at Agilent, Nina_zolotarjova@agilent.com
Liang-Sheng Yang is a manufacturing/quality engineer at AgilentLiangfirstname.lastname@example.org
Cory Szafranski is a product manager at Agilent, Cory_szafranski@agilent.com
Gordon Nicol is an R&D scientist at Agilent, Gordon_nicol@agilent.com
1. Bailey J, Zhang K, Zolotarjova N, Nicol G, Szafranski C. Removing high-abundance proteins from serum, Genetic Engineering News 2003 Nov.; 23 (19):32-37.
2. Grant SF, Kristjansdottir H, Steinsson K, Blondal T, Yuryev A, Stefansson K, Gulcher JR. Long PCR detection of the C4A
null allele in B8-C4AQ0-C4±-DR3, J. Immunol. Methods 2000; 244:41-7.