On-Line LC−MS Approach Combining Collision-Induced Dissociation (CID), Electron-Transfer Dissociation (ETD), and CID of an Isolated Charge-Reduced Species for the Trace-Level Characterization of Proteins with Post-Translational Modifications

Abstract

We have expanded our recent on-line LC-MS platform for large peptide analysis to combine collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced (CRCID) species derived from ETD to determine sites of phosphorylation and glycosylation modifications, as well as the sequence of large peptide fragments (i.e., 2000-10,000 Da) from complex proteins, such as beta-casein, epidermal growth factor receptor (EGFR), and tissue plasminogen activator (t-PA) at the low femtomol level. The incorporation of an additional CID activation step for a charge-reduced species, isolated from ETD fragment ions, improved ETD fragmentation when precursor ions with high m/z (approximately >1000) were automatically selected for fragmentation. Specifically, the identification of the exact phosphorylation sites was strengthened by the extensive coverage of the peptide sequence with a near-continuous product ion series. The identification of N-linked glycosylation sites in EGFR and an O-linked glycosylation site in t-PA were also improved through the enhanced identification of the peptide backbone sequence of the glycosylated precursors. The new strategy is a good starting survey scan to characterize enzymatic peptide mixtures over a broad range of masses using LC-MS with data-dependent acquisition, as the three activation steps can provide complementary information to each other. In general, large peptides can be extensively characterized by the ETD and CRCID steps, including sites of modification from the generated, near-continuous product ion series, supplemented by the CID-MS2 step. At the same time, small peptides (e.g., <or=2+ ions), which lack extensive ETD or CRCID fragmentation, can be characterized by the CID-MS2 step. A more targeted approach can then be followed in subsequent LC-MS runs to obtain additional information, if needed. Overall, the recently introduced ETD not only provides useful structural information, but also enhances the confidence of all assignments. The sensitivity of this new approach on the chromatographic time scale is similar to the previous Extended Range Proteomic Analysis (ERPA) using CID-MS2 and CID-MS3. The new LC-MS platform can be anticipated to be a useful approach for the comprehensive characterization of complex proteins.

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…By optimizing fragmentation energy to release O-GlcNAc (m/z 204.1), O-GlcNAc-modified peptides can be detected by ESI-MS, which has been employed in mapping O-GlcNAcylation sites in human cytomegalovirus tegument basic phosphoprotein (UL32) to serine 921 and serine 952 (Greis et al 1994). In contrast to CID, electron capture dissociation and electron transfer dissociation (ETD) are alternative fragmentation methods used to preserve more labile modifications such as phosphorylation, methylation, acetylation, glycosylation, nitrosylation, and sulfation, and allow for direct mapping of peptide/protein modifications (Mikesh et al 2006;Syka et al 2004;Udeshi et al 2007Udeshi et al , 2008Wu et al 2007;Wang et al 2010a;Sobott et al 2009). ETD fragmentation cleaves along the peptide backbone between the Cα-N producing c and z ions while still maintaining peptide side chains and modifications (Syka et al 2004;Sobott et al 2009).…”

Clustered O-Glycans of IgA1

“…For ETD LTQ XL MS/MS experiments of off-line fractionated IgA1 HR glycopeptides, a range of ETD parameters was tested to optimize fragmentation, and the data were compared with results derived from AI-ECD LTQ FT MS/MS for the same IgA1 HR O-glycoforms. Varied parameters included inclusion of supplemental activation (SA) or not, inclusion of CID of the charge-reduced species (34), and settings for the anion (fluoranthene) injection time (50 -500 ms). AGC was set to 1 ϫ 10 4 for analyte cations and 1.5 ϫ 10 5 for fluoranthene anions.…”

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

“…ETD reagent anions from fluoranthene were generated in a chemical ionization source (reagent gas: CH 4 ) and injected into the ion trap to react with the stored ionized peptides under defined conditions: 100-to 120-ms reaction time for electron transfer, active ion counting mode (ICC), and no additional delay between ETD and CRCID events. The principal difference between typically used automated CRCID (also known as supplemental activation, smart decomposition, or postactivation; subsequently referred to as autoCRCID) and the manual CRCID performed here is characterized by the ability to vary the amplitude of the activation voltage pulse and to isolate singly charged radical species before ion activation, in contrast to multiply charged radical species isolation already used in proteomics [35]. The mass spectra were acquired with a 1-min acquisition period and represent averaging over close to 100 scans; data analysis was carried out using Bruker's Data Analysis (version 3.4) software (Bruker Daltonics).…”