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. 2008 Dec 1;80(23):9034-41.
doi: 10.1021/ac801417d.

Enhanced digestion efficiency, peptide ionization efficiency, and sequence resolution for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry

Hui-Min Zhang  1 , Sasa Kazazic, Tanner M Schaub, Jeremiah D Tipton, Mark R Emmett, Alan G Marshall
Affiliations

Enhanced digestion efficiency, peptide ionization efficiency, and sequence resolution for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry

Hui-Min Zhang et al. Anal Chem. .

Abstract

Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry offers a rapid method to study protein conformations and protein-protein interactions. Pepsin is usually used to digest proteins in HDX and is known for lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FTICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatly enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein-protein interactions.

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Figures

Figure 1
Figure 1
Myoglobin fragments following separate 2 min digestions by pepsin (∼1.4 mg/mL), protease type XIII (∼1.2 mg/mL), or protease type XVIII (∼2.0 mg/mL). Fragments were desalted and separated with a Jupiter™ C5 column. The structural units of myoglobin are represented above the primary sequence (same for Figure 2).
Figure 2
Figure 2
Myoglobin peptide maps from separate digestion by pepsin (∼1.4 mg/mL) and protease type XIII (∼1.2 mg/mL). Fragments were desalted and separated with a ProZap™ C18 column. Retention time was reduced to 1.5 min (compared to 4.5 min with a Jupiter™ C5 column).
Figure 3
Figure 3
Pepsin (top) and protease type XIII (bottom) cleavage preferences for myoglobin, IPMS, RAGE and c-Kit proteins. The Preference Ratio is the number of fragments with the specified C-terminal amino acid divided by the total number of that specific amino acid in the protein.
Figure 4
Figure 4
Digestion with both pepsin and protease type XIII to increase sequence resolution within myoglobin segment 32-55. Top: HDX rate constant distribution based on maximum entropy method fit to the deuterium incorporation vs. time profile for the protease type XIII digest fragment 43-54. Bottom: Assigned slow-, intermediate-, and fast-exchanging amide hydrogens for seven peptides spanning positions 32-55. Amide hydrogens from the loop region 44-50 are shown in red. The numbers of fast- (F), medium- (M) and slow- (S) exchanging hydrogens are listed in line 9 (see text). Amino acids 40L, 41E, 42K, and 43F were identified as slow-exchanging hydrogens (blue). Of the 2 medium-exchanging hydrogens, one was narrowed down to segment 38-39 (gold), and another to amino acid 55M (gold). Four fast-exchanging hydrogens could be narrowed down to segment 44-48 (orange) and one another to segment 49-54 (light orange).
Figure 5
Figure 5
MEM assigned exchange rates for increased sequence resolution for myoglobin segment 69-93. Amide hydrogens from the loop region 78-85 are shown in red (see the inserted myoglobin 3-D structure). From the exchange rates of the 11 overlapping fragments, we assigned the number of fast- (F), medium- (M) and slow- (S) exchanging hydrogens to line 13 (see text). The amide hydrogen on amino acid 85E (in red) was identified as fast-exchanging. Three fast-exchanging hydrogens were narrowed down to segment 79-84 (in orange) and another to either 86L or 87K (orange). Of the two assigned medium-exchanging hydrogens, one is on either 86L or 87K, and another on segment 89-93 (gold). All of the backbone amide hydrogens on segment 70-77 (blue) were slow-exchanging hydrogens.
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