Purpose: The value of implementing Mass Spectrometry to analyze large molecule proteins has been broadly recognized. However, the utilization of LC-MS/MS in the quantitation of endogenous proteins or biomarkers is still in its infant status. Human IgG is the most abundant immunoglobin in the serum, with a concentration of ~10 mg/mL detected in adults. IgG deficiency is a severe health problem in the clinic, with many ongoing studies for its accurate quantitation. Current quantitation methods often suffer from either low selectivity, less specificity or narrow dynamic range. Among different approaches for bioanalysis of large molecules, the bottom-up (signature peptide identification) and top-down (intact protein analysis) are the most commonly used methods. In this study, we sought to develop an LC-MS/MS strategy that is reliable and scientific sound as well as cost-effective. It is expected to provide a wide dynamic range, streamlined workflow, and quite reliable analytical performance.
Methods: In this study, we thoroughly evaluated the bottom-up procedures on different platforms: dilution, protein precipitation (PPT), immune-capture (both on magnetic beads and adsorption plates), and a specially designed nano-surface and molecular orientation limited proteolysis (nSMOL) antibody BA kit. The signature peptides were screened by Skyline in-silico digestion and MS tuning. The transitions of 593.827/699.404 for GPSVFPLAPSSK.+2y7 and 419.755/327.695 for ALPAPIEK.+2y6+2 were monitored. The sample was purified by direct dilution with surrogate matrix or captured by anti-human Fc antibody on Dynabeads or nano-resin from nSMOL kit. After trypsin digestion, the peptides were analyzed on tandem Mass Spec Sciex 6500. The high abundance of human IgG in human serum requires 2000 to 4000 times dilution to fit in our linear range. A surrogate buffer (0.1% BSA in PBS) was used to dilute the human serum. We obtained a reproducible outcome (R >0.96), and the calculated endogenous concentration of the human IgG from each platform matched up, consistent with the reported values.
Results: Different extraction methods for IgG were tested to optimize the sensitivity and inhibit matrix interference. The curve range for dilution and PPT method was both obtained at 0.1-200 µg/mL. We also tested six lots of human serum, and the levels of IgG were comparable. The PPT method eliminates the matrix effect from the human serum by reconstituting the samples with surrogate buffer after precipitation, but a high aliquot volume was needed. Thus, we evaluated the on-bead and off-bead (plate) immune-capture enrichment. The MaxiSorp plate (ThermoScientific) has less surface area and limited loading capacity. So it is not recommended for quantitation of large molecules with a high concentration in the matrix. Dynabeads M-280 streptavidin beads capture can hit a range of 0.1-40 µg/mL (Figure 1), and the loading capacity may be customized to increase by adding more Dynabeads. Moreover, a less aliquot volume is needed (10 µL for Dynabead vs. 20 µL for PPT). The anti-human Fc antibody we used in this method generated a universal peak in ALP chromatography, and it can be used as internal standards for GPS quantitation (Figure 2).
The nSMOL kit, provided by Shimadzu, was claimed to be capable of specific IgG quantitation. Our test revealed that it has pros and cons. It could asset a rapid and convenient quantitation in a standard workflow without the capture antibody development. Overall, the analytical result was acceptable (R >0.96 at curve range 1-160 µg/mL). An issue observed was one of the monitored peptides, GPS, suffered from interference peaks. The economic concern is also considerable. We suggest it may be used for fast screening experiments, particularly for abundant large molecule proteins.
Conclusion: In conclusion, our current hybrid LC-MS/MS strategy enables us to conduct human IgG quantitation under a strict regulation scenario confidently. The easily customized dynamic range, the streamlined workflow, and quite reliable analytical performance support the on-bead-capture the most promising approach. The experience gained from this work also benefits the other large protein quantitation in a bottom-up fashion.
Xun Liu– Plainsboro, New Jersey
Xun Liu– Plainsboro, New Jersey
Alfred Simmons– Plainsboro, New Jersey
Jingyi Zhang– Plainsboro, New Jersey
Roger Demers– Plainsboro, New Jersey
Nigel Ewing– Marlborough, Massachusetts
Scott Cassidy– Somerset, Massachusetts
Ze Li– Plainsboro, New Jersey