Obtaining complementary polypeptide sequence information from a single precursor ion packet via sequential ion mobility-resolved electron transfer and vibrational activation

Abstract

Tandem mass spectrometry (MS/MS) is now well-known as a powerful tool for characterizing the primary structures of peptides and proteins; however, in many cases the use of but a single dissociation method provides only a partial view of the amino acid sequences and post-translational modification patterns of polypeptides. While the application of multiple fragmentation methods can be more informative, this introduces the burden of acquiring multiple MS/MS spectra per analyte, thus reducing the effective duty cycle of such methods. In this work, initial proof-of-concept is provided for a method designed to overcome these barriers. This method relies on the complementary fragmentation information that can be provided by performing collision-induced dissociation (CID) and electron transfer dissociation (ETD) in concert, while also taking advantage of an ion mobility (IM) dimension to temporally resolve the occurrence of CID and ETD when applied to a single accumulated packet of precursor ions. In this way, the significant proportion of the precursor ion population that remains unreacted in ETD experiments is subjected to CID rather than being fruitlessly discarded. In addition, the two distinct fragmentation spectra can be extracted from their corresponding IM domains to render readily interpretable individual fragmentation spectra. This scheme was demonstrated for several polypeptides ranging from 1.3 to 8.6 kDa in molecular weight. In each case, IM-resolved CID and ETD events resulted in b/y and c/z ions, respectively, which each covered both unique and overlapping sequence information. These findings demonstrate that the combination of CID and ETD can be achieved with greater utilization of the available ion population and little or no loss of duty cycle.

Parallel Determination of Polypeptide and Oligosaccharide Connectivities by Energy-Resolved Collison-Induced Dissociation of Protonated O-Glycopeptides Derived from Nonspecific Proteolysis

“…All such measurements were carried out using a Synapt G2-S HDMS quadrupole time-of-flight (Q-TOF) MS instrument (Waters; Manchester, UK). The instrument was fitted with a home-built static nanoflow electrospray ionization (nESI) source on which samples were delivered from a pulled glass emitter situated such that the analyte solution was in contact with a platinum wire. , The nESI emitters were prepared from borosilicate melting point capillary tubes (1.5–1.8 mm inner diameter 100 mm; (Corning Pyrex; Corning, NY) with the aid of a Flaming/Brown horizontal micropipette puller (Sutter Instruments; Novato, CA). Ionization was conducted by applying a capillary potential of 1.2–1.4 kV to the platinum wire.…”

The Role of Electron Transfer Dissociation in Modern Proteomics

“…Collisional activation during ETD reactions is a nonviable supplemental activation approach because it prevents sufficient overlap of the cation and anion clouds, which is required for the ion–ion reaction to occur . Several iterations of post-ETD activation have been explored, − with the most widely adopted being gentle resonant excitation of ETnoD products, a process termed ETcaD . Most recently, the Heck group introduced EThcD, which involves broadband activation of all ETD products with high-energy collisional dissociation (HCD) .…”

Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase

“…The limitations of CID are magnified in the case of membrane protein complexes for which typically all available collisional activation has to be used to remove the micelle, rendering it impossible to fragment the detergent-free complex and elucidate its stoichiometry . This means that the ideal of reverse-engineering protein complexes by CID, probing all levels of structure through several steps of activation, is a frontier challenge. ,, As a result, there is an urgent need to develop orthogonal means for activating protein assemblies that are unencumbered by the limitations inherent to the mass spectrometer and charge on the ions.…”

“…11 This means that the ideal of reverse-engineering protein complexes by CID, probing all levels of structure through several steps of activation, is a frontier challenge. 17,20,21 As a result, there is an urgent need to develop orthogonal means for activating protein assemblies that are unencumbered by the limitations inherent to the mass spectrometer and charge on the ions. Infrared multiphoton dissociation (IRMPD) employs IR lasers external to the mass spectrometer and is an attractive alternative to CID as the amount of energy obtained by the ions during IR activation does not depend on their charge states and can be increased by raising the intensity of the radiation without compromising ion transfer and inducing ion losses.…”