Top-Down Characterization of Proteins with Intact Disulfide Bonds Using Activated-Ion Electron Transfer Dissociation

Abstract

Here we report the fragmentation of disulfide linked intact proteins using activated-ion electron transfer dissociation (AI-ETD) for top-down protein characterization. This fragmentation method is then compared to the alternative methods of beam-type collisional activation (HCD), electron transfer dissociation (ETD), and electron transfer and higher-energy collision dissociation (EThcD). We analyzed multiple precursor charge states of the protein standards bovine insulin, α-lactalbumin, lysozyme, β-lactoglobulin, and trypsin inhibitor. In all cases, we found that AI-ETD provides a boost in protein sequence coverage information and the generation of fragment ions from within regions enclosed by disulfide bonds. AI-ETD shows the largest improvement over the other techniques when analyzing highly disulfide linked and low charge density precursor ions. This substantial improvement is attributed to the concurrent irradiation of the gas phase ions while the electron-transfer reaction is taking place, mitigating nondissociative electron transfer, helping unfold the gas phase protein during the electron transfer event, and preventing disulfide bond reformation. We also show that AI-ETD is able to yield comparable sequence coverage information when disulfide bonds are left intact relative to proteins that have been reduced and alkylated. This work demonstrates that AI-ETD is an effective fragmentation method for the analysis of proteins with intact disulfide bonds, dramatically enhancing sequence ion generation and total sequence coverage compared to HCD and ETD.

Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

“…Activated-ion electron transfer dissociation (AI-ETD) has rapidly developed as a highly effective tandem MS approach for proteomic applications. [33][34][35][36][37][38][39][40][41] The method uses concurrent infrared (IR) photoactivation to improve dissociation efficiencies and increase product ion generation in electron transfer dissociation (ETD) reactions. 11,42 We report here that the combination of simultaneous vibrational activation from IR photon bombardment and electron-driven dissociation via ETD is particularly well-suited for intact glycopeptide fragmentation.…”

“…11,12,[21][22][23][24][25][26][13][14][15][16][17][18][19][20] Even with these methods, large-scale analysis of intact glycopeptides remains largely limited to fewer than ~1,000 unique glycosite identifications from any one system or tissue (approximately an order of magnitude behind other PTMs), [27][28][29][30][31][32] and few studies assess heterogeneity across the glycoproteome with site-specific resolution.Activated-ion electron transfer dissociation (AI-ETD) has rapidly developed as a highly effective tandem MS approach for proteomic applications. [33][34][35][36][37][38][39][40][41] The method uses concurrent infrared (IR) photoactivation to improve dissociation efficiencies and increase product ion generation in electron transfer dissociation (ETD) reactions. 11,42 We report here that the combination of simultaneous vibrational activation from IR photon bombardment and electron-driven dissociation via ETD is particularly well-suited for intact glycopeptide fragmentation.…”

Characterization of the T4 gp32–ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry

“…S10b and c † ), consistent with previous reports indicating that the presence of disulfide bonds impedes fragmentation of intact proteins. 77 The ion signal originally dispersed among oxidized and reduced proteoforms may be concentrated into the reduced form after addition of the reducing agent, thus increasing the abundance of the precursor ion available for MS/MS analysis. This “concentration” of the precursor into a more homogeneous form likely contributes to the increased sequence coverage observed for the reduced protein compared to the non-reduced protein.…”

Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD

“…Although digestion below pH 7 will reduce disulfide scrambling, it has been demonstrated that significantly lower pH is required to eliminate all scrambling in antibodies. 46,21 Also, while IgGs should not contain free cysteines, non-zero levels have been detected in all four sub-classes, which can have deleterious effects on mAb function. 47 The choice of pH and protease is crucial during digestion as free cysteine is reactive down to pH 4.…”

“…Several groups have shown that electron-transfer and electron-capture dissociation (ETD/ECD) provide not only sequence information, but also readily cleave S-S bonds, leading to observation of individual peptide pairs and higher confidence in assignments. 17–18,19,20,21 Downsides to electron-based dissociation include lengthy activation time, ion charge state dependence, and low dissociation efficiency (i.e. electron transfer/capture with no dissociation is common and sometimes requires another activation stage), although significant progress towards correcting many of these shortcomings has been made.…”