Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

Abstract

Covalent labeling and mass spectrometry are seeing increased used together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g. diethylpyrocarbonate) and non-specific (e.g. hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues, and thus protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g. 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g. microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. As compared to typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 Å to 7 Å for myoglobin, 13 Å to 10 Å for cytochrome c, and 9 Å to 8 Å for β-2-microglobulin.

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules

“…These modification conditions have been established by numerous previous studies of DEPC labeling of β2m by our group. 19,21-23 …”

“…DEPC provides good structural coverage as it can react with up to 30% of the residues in the average protein, providing an effective resolution around 8-10 Å. 23 While this level of structural detail can often help define protein‐protein interaction sites, this level of resolution may not be sufficient for identifying small molecule binding sites. Consequently, we are exploring the combination of information obtained from DEPC labeling with information from other labeling reagents.…”

Enzymolysis kinetics, thermodynamics and model of porcine cerebral protein with single-frequency countercurrent and pulsed ultrasound-assisted processing

“…DS represents the variation in the extent of local disordering between transition state and the ground state. The decrease in the DS can be attributed to the oxidative modification of amino acid residues and initiation of crosslinking and aggregation which leads to the increase in enzymolysis activity [42]. DG had little change for ultrasonic treatment.…”

Label Scrambling During CID of Covalently Labeled Peptide Ions

“…We have recently shown that diethylpyrocarbonate (DEPC) is a promising reagent molecule because of its ability to modify a wide range of amino acids [1, 10-14]. This potential to modify numerous amino acids enables DEPC to probe approximately 30% of the average protein [14]. Usually, proteins that are labeled with DEPC are then subjected to proteolysis so that the modification sites can be pinpointed to individual residues, thereby improving the resolution of this method.…”