5 research outputs found
A Novel and Cost Effective Method of Removing Excess Albumin from Plasma/Serum Samples and Its Impacts on LC-MS/MS Bioanalysis of Therapeutic Proteins
We
have developed an innovative method to remove albumin from plasma/serum
samples for the LC-MS/MS quantitation of therapeutic proteins. Different
combinations of organic solvents and acids were screened for their
ability to remove albumin from plasma and serum samples. Removal efficiency
was monitored by two signature peptides (QTAÂLVEÂLVK and
LVNÂEVTÂEFÂAK) from albumin. Isopropanol with 1.0%
trichloroacetic acid was found to be the most effective combination
to remove albumin while retaining the protein of interest. Our approach
was compared with a commercial albumin depletion kit on both efficiency
of albumin removal and recovery of target proteins. We have demonstrated
that our approach can remove 95% of the total albumin in human plasma
samples while retaining close to 100% for two of three therapeutic
proteins tested, with the third one at 60–80%. The commercial
kit removed 98% of albumin but suffered at least 50% recovery loss
for all therapeutic proteins when compared to our approach. Using
BMS-C as a probe compound, the incorporation of the albumin removal
approach has improved both assay sensitivity and ruggedness, compared
to the whole plasma protein digestion approach alone. An LC-MS/MS
method was developed and validated based on this new approach for
the analysis of BMS-C in monkey serum. This assay was successfully
applied to a toxicological study. When the albumin removal method
was used in another clinical LC-MS/MS method, the sensitivity improved
10-fold to 50 ng/mL LLOQ comparing to a typical pellet digestion method
Amino acid variant in the kinase binding domain of dual-specific A kinase-anchoring protein 2: A disease susceptibility polymorphism
The focus of human genetics in recent years has shifted toward identifying genes that are involved in the development of common diseases such as cancer, diabetes, cardiovascular diseases, and Alzheimer's disease. Because many complex diseases are late-onset, the frequencies of disease susceptibility alleles are expected to decrease in the healthy elderly individuals of the population at large because of their contribution to disease morbidity and/or mortality. To test this assumption, we compared allele frequencies of 6,500 single-nucleotide polymorphisms (SNPs) located in ≈5,000 genes between DNA pools of age-stratified healthy, European-American individuals. A SNP that results in an amino acid change from Ile to Val in the dual-specific A kinase-anchoring protein 2 (d-AKAP2) gene, showed the strongest correlation with age. Subsequent analysis of an independent sample indicated that the Val variant was associated with a statistically significant decrease in the length of the electrocardiogram PR interval. The Ile/Val SNP is located in the A-kinase-binding domain. An in vitro binding assay revealed that the Ile variant bound ≈3-fold weaker to the protein kinase A (PKA)-RIα isoform than the Val variant. This decreased affinity resulted in alterations in the subcellular distribution of the recombinantly expressed PKA-RIα isoform. Our study suggests that alterations in PKA-RIα subcellular localization caused by variation in d-AKAP2 may have a negative health prognosis in the aging population, which may be related to cardiac dysfunction. Age-stratified samples appear to be useful for screening SNPs to identify functional gene variants that have an impact on health