299899-45-9Relevant articles and documents
Guanidinated protein internal standard for immunoaffinity-liquid chromatography/tandem mass spectrometry quantitation of protein therapeutics
Yang, Wenchu,Kernstock, Robert,Simmons, Neal,Alak, Ala
, p. 1489 - 1500 (2014/06/10)
RATIONALEA protein internal standard (IS) is essential and superior to a peptide IS to achieve reproducible results in the quantitation of protein therapeutics using immunoaffinity-liquid chromatography/tandem mass spectrometry (LC/MS/MS). Guanidination has been used as a protein post-modification technique for more than half a century. A decade ago, the modification was applied to lysine-ending peptides to enhance their MALDI responses and peptide sequencing coverage. However, rarely has tryptic digestion of guanidinated proteins been investigated, likely due to the early conclusion that trypsin did not hydrolyze peptide bonds involving homoarginine in guanidinated proteins. In this study, the opposite was observed. Guanidinated lysine residues of proteins did not hinder the access of trypsin allowing for proteolytic digestion. Based on this observation, a new concept of internal standard, named Guanidinated Protein Internal Standard (GP-IS), was proposed for LC/MS/MS quantitation of protein therapeutics. METHODSThe GP-IS is prepared by treating a portion of the therapeutic protein (analyte) with guanidine to convert arginine residues in the protein into homoarginine residues. After tryptic digestion, the GP-IS produces a series of homoarginine-ending peptides plus another series of arginine-ending peptides. One of the homoarginine-ending peptides, which corresponds to the analyte surrogate (lysine-ending) peptide, was chosen as a peptide internal standard (GP-PIS) for LC/MS/MS quantitation. RESULTSUsing this GP-IS approach, a sensitive and robust immunoaffinity-LC/MS/MS assay was developed and fully validated with a linearity range from 10 to 1000 ng/mL using 200 μL of human serum for the quantitation of an Astellas protein drug in clinical development. CONCLUSIONSThe proposed strategy allows LC/MS/MS to play an ever-increasing role in bioanalytical support for protein therapeutics development because of its capability of completely tracking all variations from the beginning to the end of sample analysis, easier preparation compared to isotope-labeled protein-IS, and greater flexibility for changing to alternate analyte surrogate peptides. Copyright
Direct analysis of reversed-phase high-performance thin layer chromatography separated tryptic protein digests using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system
Emory, Joshua F.,Walworth, Matthew J.,Van Berkel, Gary J.,Schulz, Michael,Minarik, Susanne
experimental part, p. 21 - 33 (2010/10/21)
The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, beta-casein, lysozyme and bovine serum albumin (BSA)] were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1 M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins. US Government 2009 All rights reserved.