Using recombinant DNA technology for expression of protein therapeutics is a 
maturing field of pharmaceutical research and development. As recombinant 
proteins are increasingly utilized as biotherapeutics, improved methodologies 
ensuring the characterization of post-translational modifications (PTMs) are 
needed. Typically, proteins prepared for PTM analysis are proteolytically 
digested and analyzed by mass spectrometry. To ensure full coverage of the PTMs 
on a given protein, one must obtain complete sequence coverage of the protein, 
which is often quite challenging. The objective of the research described here 
is to design a protocol that maximizes protein sequence coverage and enables 
detection of post-translational modifications, specifically N-linked 
glycosylation. To achieve this objective, a highly efficient proteolytic digest 
protocol using trypsin was designed by comparing the relative merits of 
denaturing agents (urea and Rapigest SF), reducing agents [dithiothreitol (DTT) 
and tris(2-carboxyethyl)phophine (TCEP)], and various concentrations of 
alkylating agent [iodoacetamide (IAM)]. After analysis of human apo-transferrin 
using various protease digestion protocols, ideal conditions were determined to 
contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for 
alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. 
This method was successfully applied to a novel recombinant protein, human lysyl 
oxidase-like 2. Furthermore, the glycosylation PTMs were readily detected at two 
glycosylation sites in the protein. These digestion conditions were specifically 
designed for PTM analysis of recombinant proteins and biotherapeutics, and the 
work described herein fills an unmet need in the growing field of 
biopharmaceutical analysis.