The Use of UVPD coupled with Ion Mobility Mass Spectrometry to determine structure and sequence from drift time selected peptides and proteins
Alina Theisen1, Bin Yan1, JefferyM.Brown2, Michael Morris2,Bruno Bellina1*, Perdita E. Barran1*
1Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, & Photon Science Insitute, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
2Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, UK.
*Email for Correspondence: , ,
Abstract
We demonstrate the capabilities of a laser-coupled ion mobility mass spectrometer for analysis of peptide sequence and structure showing UVPD spectra of mass and mobility selected ions. A Synapt G2-S mass spectrometer has been modified to allow photointeraction of ions post the mobility cell. For this work we have employed a single wavelength laser, which irradiates at 266nm.We present the unique capabilities of this instrument and demonstrate several key features. Irradiation of LHRH, GHRP-6 and TrpCage yields extensive b and y- type fragmentation as well as a-and c-type ions. In addition we observe side chains losses, including the indole group from tryptophan,and immonium ions. For negatively charged ions we show the advantage of using CID post UVPD: radical ions are produced following irradiation and these fragment with higher efficiency. Further, we have incorporated ion mobility and subsequent drift time gating into the UVPD method allowing the separate analysis of m/z-coincident species, both conformers and multimers. To demonstrate we selectively dissociate the singly charged dimer or doubly charged monomer ofthe peptide gramicidin A, and conformers of the [M+5H]5+ form of the peptide melittin. Each mobility selected form has a different ‘fingerprint’ dissociation spectrum, both predominantly containing b and y fragments. Differences in the intensities of various loss channels between the two species were revealed. The smaller conformer of melittin has less cleavage sites along the peptide backbone than the larger conformer suggesting considerable structural differences. For Gramicidin single laser shot UVPD discriminates between primary photodissociation and subsequent fragmentation of fragments.We also show how this modified instrument facilitates activated electron photodissociation. UVPD-IM-MS analysisservesboth as a method for peptide sequencing for peptides of similar (or identical) m/z and a method for optical analysis of mobility separated species.
Introduction
Mass spectrometry is commonly used for analysis of complex mixtures and identification of proteins and peptides, making it acentral technology in the field of proteomics1,2. In many proteomic experiments, fragmentation of mass selected peptide ions is used to identify proteins with reference to genomic databases.Many ion activation techniques have been developed over the past 30 years, to provide better sequence coverage and/or more rapid analysis3,4. For proteomic applications fragmentation data should be reproducibleas well asdiagnostic of the primary sequence, this can also be used to provide information on the three dimensional structure or conformation of the peptide or protein under examination5,6.Whilst the most commonly used fragmentation method, collision-induced dissociation (CID), is implemented on all commercially available tandem mass spectrometers7, it has some drawbacks. These are predominantly a result of the mode of energy deposition and preferential cleavage of the weakest bond, manifesting in loss of post-translational modifications, non-covalent interactions and at times limited sequence coverage8.Electron-based methods such as electron-capture dissociation (ECD) or electron-transfer dissociation (ETD), whilst usually less efficient, are often complementary to CID and as such can overcome some of its limitations4,9. While these electron-based methods allow retention of post-translational modifications (PTMs) and produce greatly enlightening fragmentation patterns, they are highly dependent on the charge density of the analyte ion and only applicable on some instruments7.
The coupling ofmass spectrometry with laser systems has long been available, and is used widely for spectroscopic investigation where mass separation is required. Many laser-coupled MS applications use photodissociation to interrogate the ion, which can take place if ions and photons are co-located long enough for absorption of one or more photons to occur1. Fragmentation then proceeds via two pathways; either the internal energy introduced into the ion by absorption of photons is converted into vibrational modes and distributed by intramolecular vibrational relaxation and fragmentation then occurs in the ground state, or dissociation occurs directly in the excited electronic state1,10. The first mechanism produces CID-like fragmentationand is accessed in infrared multiphoton dissociation (IRMPD) which requires absorption of multiple or many photons11, while the second one is usually attributed to short-wavelength photons (less than 200 nm) and produces fragments due single photon processes1. By adjusting the wavelength, ion exposure time and laser power, the energy deposited into the ion can be modulated, and this approach is gaining popularity in application to peptide and protein analysis12.
Fragmentation at longer wavelengths (200-400 nm) requires the presence of chromophores with suitable absorption cross sections; for example with a fixed wavelength laser at 266nm, dissociation of a peptide will only occur if the sequence contains aromatic amino acids (tyrosine, tryptophan, phenylalanine)1, although this wavelength has also been shown to be capable of cleaving disulphide bonds leaving two radical ions13,14. The need for suitable chromophores is lifted at 193nm and below as the backbone amide group begins to absorb8. At this wavelength, a single photon deposits enough energy into a protein ion to cause fragmentation irrespective of sequence or length, making it an amenabletechnique for top-down analysis of intact proteins13. Unlike CID which exhibits better sequence coverage at the termini of proteins, ultraviolet photodissociation (UVPD)can produce cleavage throughout13. Since it is not necessarily the most labile bonds that are broken first, UVPD allows retention of post-translational modifications13. Another advantageous characteristic of UVPD is the ability to dissociate side-chains which allows discrimination between leucine and isoleucine1,15. Overall, PD is well suited to application in proteomics and an increasing number ofinstruments have been modified to implement photodissociation16–24.A notable example of thisis inthe recent work by the Brodbelt group who demonstrated UVPD at 193 nm of Myoglobinand related fragmentation yields to the flexibility or rigidity of the structural elements that cleavage occurred at25. Reilly and co-workers used 157 nm photons to investigate fragmentation of proline-containing peptides and this allowed discrimination between the two conformations of proline even when multiple prolines were present in the sequence26. This has since been extended to whole proteins with the example of Ubiquitin, for which specific fragmentation depending on proline isomerisation was observed12.
Mass spectrometry alone is unable to distinguish between isobaric species; however this can be achieved by incorporation of ion mobility as an orthogonal method of separation. Typically, IMS involves pulsing ions into a chamber filled with neutral buffer gas to which a weak electric field is applied. Ions are pulled through this chamber by the field but are retarded by collisions with the buffer gas. The time it takes an ion to reach the end of the drift tube, that is its mobility, is dependent on its physical characteristics such as size and charge. In travelling-wave ion mobility (TWIMS), ions are moved through a buffer gas filled series of stacked ring electrodes by a wave-like DC potential27. This technique allows the separation of species that populate the same m/z such as mass-coincident monomer and dimer, as well as of molecules that adopt a variety of different conformations.
In order to explore and further develop the application of laser-coupled MS to the area of structural research, wehave modified a TWIMS-enabled Q-ToF mass spectrometer to enable photodissociation within the instrument17. While we previously demonstrated UVPD and mobility selection in this setup using a modelsystem (Flavin Mononucleotide), here we report enhanced capabilities of the instrument using a variety of peptide systems including photodissociation of both mass and conformer-selected ions.
Experimental Section
Materials
HPLC-grade water and methanol (>99.9% purity) were purchased from Sigma Aldrich (UK).Gramicidin A (sequence HCO-VGALAVVVWLWLWLW-NHCH2CH2OH) and luteinizing hormone releasing hormone [D-Trp6]-LH-RH (sequence pE-HWSYWLAPG-NH2, also known as LHRH or GnRH) were purchased from Sigma Aldrich with a purity of 90% and 99% respectively. Growth hormone releasing hexapeptide GHRP-6 (sequence HWAWFK-NH2) was purchased from GeneCust (Luxembourg) as a lyophilised powder with a purity of 99%. Melittin from honey bee venom (sequence GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) was purchased from Sigma Aldrich (UK) with a purity of 97%.TrpCage (sequence NLYIQWLKDGGPSSGRPPPS) was synthesized by FMOC solid state peptide synthesis, purified and lyophilised.
Gramicidin A, GHRP-6 and LHRHwereprepared in 50/50 water/methanol to a peptide concentration of 50 µM while Melittin was prepared to a concentration of 20 µM. TrpCage was dissolved in 50 mM ammonium acetate to a peptide concentration of 50 µM.
Ion Mobility Mass Spectrometer - achieving UVPD on m/z and conformer selected ions
Several modifications have been made to a Waters Synapt G2-S to permit injection of a laser beam23into the transfer cell, post mobility separation. A CaF2window has been incorporated into the upper vacuum flange of the time of flight region. The push plate assembly has been machined to accommodate a custom size mirror (12.7 x 5 x 1.52 mm) coated with UV-enhanced aluminium (Thorlabs) mounted at a 45 degree angle to guide the laser beam collinear to the ion beam through the entire setup. In order to facilitate laser entrance into the transfer cell, the internal diameter of the transfer cell’s exit plate has been increased from 2 mm to 3.3 mm. Finally, to perform selection of ions based on their mobilities, the exit plate of the mobility cell is grounded. This plate is then acting as a defocusing lens allowing only a defined drift-time window to enter the transfer cell region.17
The instrument’s software (MassLynx) and the Waters Research Enabled Software (WREnS) are used in tandem to achieve ion transmission through the instrument as well as trapping and conformer selection prior to detection. WREnS controls ion trapping and extraction to the time-of-flight by applying sequences of DC potentials to the stacked ring electrodes of the TWIMS region (SI Scheme S1). The look-up table function of MassLynx is used to define the drift-time window of the chosen conformer. An electromechanical shutter triggered by the WREnS trapping signal via an Arduino Uno board28 synchronizes laser irradiation with trapping of the ions.
Alternatively to the trapping approach, conformer-selective UVPD has been carried out by using the ion mobility cycle to trigger laser irradiation. A Stanford delay generator is used to match the laser activation with the drift time of a specific conformer (SI Scheme S2). The location within the TWIMS region where photodissociation occurs can be varied. Due to the collinear geometry of laser beam and ion beam, a delay of 0 equals photodissociation in the trap cell/at the very beginning of the IMS cell before separation occurs, whereas setting the delay to the drift time of a certain conformation means photodissociation of this molecule occurs in the transfer cell after ion mobility separation has taken place.
Experimental Workflow
Samples were ionized using a nanoESI source in positive ion mode with a capillary voltage of in the range of 1.2kV, sampling cone ranging from 30V to 80 V and a source temperature of 25˚C. The desired species was mass selected using the quadrupole, trapped in the transfer cell and activated using the UV laser. Optionally, specific conformations were selected by imposing a gate at the end of the mobility cell defocusing ions other than those arriving in the chosen drift time window. Typical trap fill times were 200 ms, typical activation times ranged from 500 ms to 1 s. Ions were irradiated with a Q-switched, Nd:YAG Continuum Minilite II operating at a wavelength of 266 nm and a repetition rate of 10 Hz.The average pulse energy was measured at 1 mJ.Typical acquisition times for the spectra shown here ranged from 10 minutes to a maximum of 1 hour, although data of sufficient intensity,depending on the analyte and chromophore, can be obtained in 20 seconds which would be compatible with an HPLC workflow.Data was analysed using MassLynx v4.1 (Waters Corporation, USA), OriginPro 9.1 (OriginLab Corporation, USA) and Microsoft Excel 2010 (Microsoft, USA).
Results and Discussion
Figure 1: UVPD at 266 nm of LHRH and GHRP-6. a) Doubly charged LHRH was trapped and irradiated for 1 second (10 laser shots), yielding a series of b and y ions as well as a, c and x type fragments.b) Singly charged GHRP-6 was trapped and irradiatedfor 500 ms (5 laser shots). UVPD of both peptides produces a plethora of fragments, of which the dominant ions, mainly b and y, have been annotated. H refers to the histidine immonium ion while W represents the tryptophan side chain. The laser produces noise peaks in the lower m/z region which have been annotated with an asterisk*.
- UVPD of peptides and a mini protein
The peptides that we have initially examined all containthe aromatic residues tryptophan (W) and tyrosine Y and are therefore amenable to UV fragmentation at 266 nm.The doubly charged monomer of a D-Trp6 analogue of Luteinizing Hormone Releasing Hormone (LHRH), calculated Mw 1311.9 Da and measured at m/z 656.5, was mass selected in the quadrupole and trapped to allow UV irradiation.The resulting UVPD spectra can be seen in Figure 1a; the CID and non-activated spectra can be found in the supplementary information(SI Figure S1).UVPD results in a spectrum with a wealth of features, and an overall fragmentation yield of 0.57 (defined as the sum of the fragment ions’ intensity divided by the intensity of all ions) compared to 0.2 when trapping without laser irradiation. Spontaneous fragmentation of trapped ions without further activation is occasionally observed mainly for multiply charged ions. This is likely to be due to a combination of collisional activation with the argon buffer gas and the presence of mobile protons29. The UVPD fragments are predominantly b and y type ions as well as some a ions and a few other loss channels. UVPD at 266nm has produced ions b2-9 and y1-9 indicating that total sequence information is contained in the spectra following cleavage at every peptide bond. In addition to these dominant b and y loss channels, loss of a small molecule (-18 Da attributed to loss of H2O) is apparent. The dissociation data is comparable to CID (SI Figure S1a) although the intensities of different loss channels vary.There are notable differences in the low m/z region, where UVPD produces the histidine immonium ion (H) at m/z 110 in comparatively high abundance. This is in accordance with previous studies, in which immonium ions from histidine, tryptophan, tyrosine and phenylalanine were found to be produced abundantly in photodissociation but only to a lesser extend in low-energy CID30,31. The histidine immonium in LHRH as an internal fragment is most likelyproduced by a combination of y- and a-type cleavage, in this case y-type cleavage of the a2 fragment also visible in the obtained spectrum30. While the a2 ion is produced by both CID and UVPD, the conversion from a2 into Happears increased by UVPD, even though the overall intensity of the a2 ion remains higher than H. Again, this has been seen previously and could be attributed to a high stability of the a2 ion32,33.
The histidine residue in LHRH has been shown to be involved in many interactions, with the C-terminus amongst others, giving the molecule a ring-like shape which the histidine side chain sits inside in34. It is remarkable that this residue which appears important for the three-dimensional structure of the ion is lost readily.
The UVPD spectrum of LHRH also reveals a small amount of [M+H]+originating from charge stripping of the precursor ion, however this is also observed without laser irradiation (SI Figure S1b) and may be due to proton loss during the trapping. UVPD displays a rather prominent peak at m/z 130 attributed to the tryptophan side chain which could be explained by it being the site of photon absorption.
Similar results are shown for GHRP-6 (Figure 1b), again the dominant fragments are b and y ions, but there is an increase in intensity of the histidine immonium ion and tryptophan side chain peakfrom the UVPD compared to CID (SI Figure S2a). The terminal histidine immonium ion in this case may have been produced by a rearrangement of the oxazolone ring of the corresponding b ion30. Compared to LHRH, the immonium ion is more abundant in the spectrum of GHRP and this may be attributed to two reasons: GHRP has only 6 residues (compared to 10) and therefore has less vibrational modes available to it which could dissipate energy. Secondly, it has been shown that the closer the precursor residue is to the N-terminus, the more abundant the immonium ion30,35.
In addition to b, y and a ions, photodissociation of GHRP-6 also yields the internal sequence fragments WAW and AWF-28.Interestingly, UVPD of this peptide also shows the unique fragment v6, a result of side chain cleavage of the histidine residue, which is not found in the CID spectrum.Typically, v-type fragments are associated with fragmentation at high energies and are found in higher-energy collisional dissociation (HCD) and vacuum UVPD spectra, although they have not previously been reported with 266 nm photons to our knowledge.
Figure 2: UVPD spectrum with an irradiation time of 1 second (10 laser shots) of TrpCage. UV activation yields a full series of b and y ions as well as loss of small neutral molecules such as CO2 (-44) and H2O (-18). The inset shows the isotopic distribution at the precursor m/z for CID and UVPD, revealing an increase in [M+H]²⁺ upon laser activation (highlighted in blue).The most dominant fragments have been labelled on the spectrum.
Following the experiments on small peptides described above we then applied our UVPD methodology to a mini protein termed TrpCage36. The [M+2H]2+ ion (measured at m/z 1085.8, calculated Mw 2169.4) was mass selected, trapped and irradiated; a wealth of fragments were produced showing extensive sequence coverage of this large molecule (Figure 2). The overall fragment yield was 0.26 compared to 0.15 without irradiation, and as for the peptides above, the fragment channels are comparable to those obtained with CID with some distinction in intensities (SI Figure S3b). A notable loss channel that is significantly enhanced in UVPD is the neutral loss of 44 Da attributed to CO2.The most distinct difference between UVPD (Figure 2) and CID (SI Figure S3a) is a significant increase in the [M+H]²⁺ peak at m/z 1084.8 corresponding to the loss of hydrogen from the precursor ion. Previous studies on UVPD of amino acids and peptides have also observed this dissociation channel37,38. Sobolewski et al. explained this fragmentation channel in terms of crossing from the ππ* state to the πσ* state, transferring an electron from the aromatic ring of a residue to protonated N-H and subsequent loss of H37.