probing the gas-phase folding kinetics of peptide ions by IR activated DR-ECD

Cheng Lin 0 a Jason J. Cournoyer 0 a 0 b 0 Peter B. O'Connor 0 2 0 Address reprint requests to Dr. Peter O'Connor, Deparbnent of Biochem istry, Boston University School of Medicine , Boston, MA 02118, USA 1 Department of Chemistry, Boston University , Boston, Massachusetts, USA 2 Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine , Boston, Massachusetts, USA The effect of infrared (IR) irradiation on the electron capture dissociation (ECD) fragmentation pattern of peptide ions was investigated. IR heating increases the internal energy of the precursor ion, which often amplifies secondary fragmentation, resulting in the formation of w-type ions as well as other secondary fragments. Improved sequence coverage was observed with IR irradiation before ECD, likely due to the increased conformational heterogeneity upon IR heating, rather than faster breakdown of the initially formed product ion complex, as IR heating after ECD did not have similar effect. Although the ECD fragment ion yield of peptide ions does not typically increase with IR heating, in double resonance (DR) ECD experiments, fragment ion yield may be reduced by fast resonant ejection of the charge reduced molecular species, and becomes dependent on the folding state of the precursor ion. In this work, the fragment ion yield was monitored as a function of the delay between IR irradiation and the DR-ECD event to study the gas-phase folding kinetics of the peptide ions. Furthermore, the degree of intracomplex hydrogen transfer of the ECD fragment ion pair was used to probe the folding state of the precursor ion. Both methods gave similar refolding time constants of ~ 1.5 s-1, revealing that gaseous peptide ions often refold in less than a second, much faster than their protein counterparts. It was also found from the IR-DR-ECD study that the intramolecular H- transfer rate can be an order of magnitude higher than that of the separation of the long-lived clz product ion complexes, explaining the common observation of c and z type ions in ECD experiments. (J Am Soc Mass Spectrom 2008,19,780-789) 2008 American Society for Mass Spectrometry - O biology is to understand the process of in vivo ne of the major challenges of modem structural folding of a protein into a well-defined, biolog ically active structure [1, 2]. The effects of aqueous solvation and the intrinsic intramolecular interactions may be better revealed by studying the protein confor mation in the gas phase in the absence of solvents. Making use of the soft ionization method of electro spray ionization (ESI) [3], a number of mass spectrom etry (MS) based methods have been applied to investi gate the protein conformation in the gas phase, includ ing the ESI charge state distribution to determine the availability of ionization basic sites [4, 5], HID ex change (HDX) to identify the exposed region of the conformation [6-10], drift tube ion mobility spectrom etry (IMS) to measure the conformational cross section [10-13], high-field asymmetric waveform ion mobility spectrometry (FAIMS) to separate different conformers [10, 14, 15], infrared photodissociation spectroscopy (IRPDS) to probe the hydrogen bonding [16-20], and electron capture dissociation (ECD) [21,22] to locate the noncovalent tertiary bonding [23-26]. Particularly, ECD based methods have been used to study the gas-phase unfolding and refolding kinetics of protein ions [24, 25J. ECD in Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS or FTMS) [27, 28J has quickly found wide application in both top-down and bottom-up proteomics [21, 29-33J, as well as in identi fying and locating post-translational modifications (PTMs) [34-38J. As a nonthreshold dissociation method, the ECD fragment ion yield of a protein ion is not only affected by its sequence, but also by its higher order structures [15, 21, 26, 39, 40J. This is the basis of studying protein conformation and folding process by ECD. Noncovalent intramolecular interactions of pro tein ions may prevent fragment ion separation, leading to decreased product ion yield and poor sequence coverage, both of which can be improved in activated ion (AI)-ECD, where protein ions are unfolded [41-45]. Ion activation is typically done by collisions with back ground gas molecules [41, 43, 45J, raising the ambient temperature [16, 45], or infrared (IR) laser irradiation [42,44-46]. Efficient energy-transfer via multiple colli sions with gas molecules requires the cell pressure to be increased to the 1O~6 torr range, which in tum requires a long pump-down time (~10 s typically) before the pressure is suitable for trapped ion excitation and detection with a sufficiently long transient for good resolving power, making it unattractive for highthroughput analysis or signal averaging when the sample comes in limited amount. Moreover, collisional activation via sustained off-resonance irradiation (SORI) [47, 48] of precursor ions is complicated by tuning of optimal conditions for each individual ion, while in-beam activation (as in in-beam ECD [41] or plasma ECD experiments [43]) has the limitation of not being able to isolate the precursor ions. Raising the temperature to and keeping it at a well defined value in the ICR cell is not always possible with every FT-ICR mass spectrometer [49, 50]. Even when it is possible, it is still inconvenient in that it takes time to heat up the cell before AI-ECD experiments and to cool the cell back down for normal operation afterwards. Furthermore, heating also usually results in an undesirable increase in cell pressure. IR irradiation, on the other hand, is fast, easy to implement, allows precursor ion isolation, and does not result in a cell pressure increase, making it the preferred method for ion activation in AI-ECD experiments. Simultaneous introduction of the IR laser and electron beam to intersect the ion cloud in the center of the ICR cell has been previously done by either bringing in one beam off-axis while keeping the other on-axis [42, 45], or introducing the IR beam through a hollow electron gun mounted on-axis and letting the ringshaped electron beam be compressed by the magnetic field gradient [44]. It has been shown extensively that ion activation by IR laser absorption leads to enhanced fragmentation in protein ion ECD. The heated, unfolded protein ion can also cool and refold in the gas phase, and ECD fragment ion yield with ECD conducted at various delays after the IR irradiation should provide a measure of the extent of protein refolding [25]. It should be noted that the increased product ion formation results from the breakdown of the initially formed long-lived fragment ion complexes that stay unseparated during the excitation/detection event in the absence of ion activation. Unlike larger protein ions, intramolecular noncovalent interactions in peptide ions are not as numerous. Thus, the resulting fragment ion complexes in ECD are ofte (...truncated)