focus on Mass Spectrometry & Spectroscopy


High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) and Electron Transfer Dissociation (ETD) Mass Spectrometry for the Separation of Phosphopeptide Isomers and Analysis of Gas-Phase Phosphate Relocation.


Jade Smart*, Helen J. Cooper, Department of Biosciences, The University of Birmingham, Birmingham, UK *Recipient of a BMSS summer studentship award. Reproduced with kind permission from the British Mass Spectrometry Society newsletter, Mass Matters 67th Edition, April 2012, pages 28-30.


Phosphorylation is a reversible post-translational modification (PTM) of proteins. A phosphate group is added to a serine, threonine or tyrosine residue via a covalent bond. Phosphorylation of proteins is important in biological molecules as it plays a role in various pathways including cell signalling and metabolism, amongst other activatory or inhibitory roles. However, atypical phosphorylation can be detrimental and has been linked to cancer, Alzheimer’s disease and a variety of other illnesses. [1, 2] For these reasons it is important that the detection and localisation of phosphorylated residues is accessible.


Tandem mass spectrometry (MS/MS) allows an in depth chemical analysis of various molecules by fragmenting ions. Collision induced dissociation (CID) is a slow heating technique that fragments the precursor ion using a low energy pathway. The low energy causes the weak peptide (CO-N) bond to break, forming b and y ions. CID of phosphorylated peptides often results in the loss of either 98 Da (phosphoric acid) or combined loss of 80Da and 18Da (phosphate and water). CID therefore allows us to identify the presence of a phosphorylation within the peptide; however it often fails to locate the exact residue that is phosphorylated.


Syka et al. [3] introduced Electron Transfer Dissociation (ETD) in 2004 as an additional fragmentation technique. ETD fragmentation is similar to ECD. [4] ETD fragments multiply charged cations by transferring electrons to them and forming charge-reduced radical ions. [4] The N-Cα bond is broken forming c and z• ions, and PTMs are preserved on the backbone. [5]


This is advantageous over CID as it increases the possibility of accurately localising the PTM sites. This also means that isobaric phosphopeptides can be identified. ETD fragmentation is random and non-selective, therefore allowing for a higher peptide sequence coverage than selective processes such as CID. [6]


A problem associated with ETD is the failure to completely fragment doubly-charged ions. [6] Unlike CID, ETD does not break the non-covalent bonds, this causes improper separation of the fragments, producing large charge reduced species and spectra which contain few fragment ions. Coon et al. overcame this problem with the introduction of supplemental activation ETD; the charge reduced ion is collisionally activated which allows separation of the fragments and more informative spectra. [7] Coon et al observed that using supplemental activation increased the sequence coverage and enhanced peptide fragmentation when compared to standard ETD. [8, 9]


High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates ions in the gas phase at atmospheric pressure on the basis of their mobility at changing electric fields. [10] A voltage is applied via an asymmetric waveform causing the ions to oscillate between two electrodes. Each ion will have its own mobility towards the electrodes, however to exit from the FAIMS device the ion must be ‘balanced’ between them.


To ‘balance’ the ions, a compensation voltage (CV) is applied. Each ion has a range of CV where it is transmitted into the mass spectrometer. The CV therefore is used to define which ions are transmitted, thereby separating them. [11] FAIMS is not based solely on mass to charge ratio so it can therefore be used to separate isobaric species which is useful in phosphopeptide analysis.


Reid et al. have previously identified the gas phase relocation of a phosphate group on a peptide within CID. [12] Phosphate relocation can become a real problem to protein analysts as it leads to inaccurate localisation of the phosphorylation site.


The aim of this work is to identify the possibility of gas-phase phosphate relocation within saETD and FAIMS-ETD analysis and determine the optimum conditions in LTQ Orbitrap Velos saETD and FAIMS where this is not observed. We have varied the supplemental activation energy and the dispersion voltage applied to FAIMS and observed the differences in fragmentation of a set of 9 synthetic peptides.


Method


The following peptides were synthesised by Alta Biosciences (Birmingham, UK): 1. APLsFRGSLPKSYVK 2. APLSFRGsLPKSYVK 3. APLSFRGSLPKsYVK 4. NTNEyTEGPTVVPR 5. NTNEYtEGPTVVPR 8. APLSFLGSLPKsYVK 9. APLsFLGSLPKSYVK 11. LFtGHPESLER 12. LFTGHPEsLER


s is phosphoserine, t is phosphothreonine, y is phosphotyrosine


The peptides were re-suspended in water and made up to a 2µM concentration with a solution of 70% methanol, 30% water and 2% formic acid. Peptides were directly infused by use of an Advion Nanomate Triversa and then subject to ETD (100 ms activation 2+, 66.67 ms activation 3+) in a LTQ Orbitrap Velos (Thermo Fisher Scientific). The mass spectrometer was set to a resolution of 30,000, 1 microscan, and an AGC target of 5x104. Each peptide was analysed at varying supplemental activation energies, from 0 to 25% in graduations of 2.5.


Once suitable saETD parameters were determined, i.e., those where phosphate transfer/loss were not observed, 2µM solutions of each peptide were subject to FAIMS. We set the FAIMS at a supplemental activation of 7.5% and analysed each peptide in both the doubly and triply charged states.


The FAIMS mass spectra were recorded at 7500 resolution, AGC target of 5 x 104 automatic gain control. Each scan comprised 5 microscans. Each analysis consisted of 204 scans, each at different compensation voltages, varying from -60 V to -0.10 V in 0.30 V graduations. In the FAIMS device, we applied different dispersion voltages: -5000, -4000 and -3000 V to determine if this had any effect on the appearance of the neutral loss peaks.


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