Statistical Examination of the Emphasis Type="Ita

Authors: Lindsay J Morrison Jake A Rosenberg Jonathan P Singleton Jennifer S Brodbelt

Publish Date: 2016/05/20

Volume: 27, Issue: 9, Pages: 1443-1453

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Abstract

Dissociation of proteins and peptides by 193 nm ultraviolet photodissociation UVPD has gained momentum in proteomic studies because of the diversity of backbone fragments that are produced and subsequent unrivaled sequence coverage obtained by the approach The pathways that form the basis for the production of particular ion types are not completely understood In this study a statistical approach is used to probe hydrogen atom elimination from a + 1 radical ions and different extents of elimination are found to vary as a function of the identity of the Cterminal residue of the a product ions and the presence or absence of hydrogen bonds to the cleaved residueAdvances in high throughput bottomup proteomics approaches 1 2 have been driven in part by improvements to established ion activation methods and developments of new methods 3 4 New activation techniques such as electronbased 5 6 7 8 and photoactivation methods 9 10 11 have resulted in the production of different types and distributions of diagnostic fragment ions than ones generated by conventional collisional activation methods Ideally for sequencing applications fragmentation of peptides would be restricted to one or more of the three bonds of the peptide backbone Cα–C C–N and N–Cα leaving sidechains and modifications intact aside from those that successfully differentiate isobaric leucine and isoleucine wtype ions These are the dominant cleavages promoted by collisional electronmediated and photonbased activation for most peptides and in silicobased database search algorithms have proven successful for identification of thousands of peptides in numerous proteomics applications As a consequence of extensive studies involving examination of large populations of peptides CID fragmentation is well understood and can be explained by the mobile proton and the pathways in competition models 12 13 of which the latter largely encompasses the mobile proton model These studies have uncovered several preferential backbone cleavages typically observed at proline and aspartic acid which can be used in a predictive manner to facilitate peptide sequencing 14 15 16 17 For example upon collisional activation prolinecontaining peptides exhibit enhanced cleavage of the amide bond Nterminal to the proline residues a process that is particularly prominent for peptides in higher charge states 14 In contrast preferential cleavage of the amide bond located Cterminal to acidic residues is exaggerated for peptides in low charge states 15 16 The statistical characterization of peptide fragmentation has been pursued in a number of other collisional activation studies 18 19 20 21 The inroads in deciphering the fragmentation of peptides upon collisional activation has spurred significant interest in understanding the underpinnings of other activation processes including mechanistic aspects and the factors that contribute to variations in product ion abundances as a function of peptide length sequence and charge stateUVPD is a relatively new activation approach that has rapidly developed in part because it yields unrivaled sequence coverage in large peptides and proteins 22 23 Activation by absorption of 193 nm ultraviolet photons promotes cleavage of all three backbone bonds resulting in the formation of a and x cleavage of the Cα–C bond c and z cleavage of the N–Cα bond as well as b and y type ions In addition to these six ion types a + 1 x + 1 x + 2 and y – 2 ions are also prevalent due to various hydrogen atom migrations that can occur via radicalmediated pathways Despite the promise of 193 nm UVPD the mechanisms that govern the formation of these ions are not well understood Consequently algorithms and models that take advantage of 193 nm UVPD fragmentation are largely absent or underdevelopedThe Reilly group has presented several elegant studies that examine UVPD mechanisms using fragmentation and modeling of small model peptides 24 25 26 27 28 29 30 They have shown that activation of dipeptides by 157 nm UV radiation results in the elevation of an electron to a Rydberg orbital which causes scission of the Cα–C bond by a Norrish type I mechanism to generate a + 1 ion and x + 1 radical ions depending on the location of an arginine residue 25 26 27 Radical elimination via amide hydrogen or βhydrogen elimination was shown to account for the a ions 25 26 27 Here we use lysN digestion to generate an ion series for a large number of peptides and use statistical analysis of the abundance of the a and a + 1 ions to explore hydrogen elimination from a+1 ionsMethanol DTT iodoacetamide hemoglobin betalactoglobulin carbonic anhydrase myoglobin lysozyme albumin betacasein and kappacasein were obtained from SigmaAldrich St Louis MO USA Guinea pig adrenocorticotropic hormone ACTH 139 sequence SYSMEHFRWGKPVGKKRRPVKVYANGAEEESAEAFPLEF was obtained from Bachem Torrance CA USA KGTDVLAWIRGCRL was obtained from ABI Scientific Sterling VA Proteins lacking disulfide bonds were digested by incubation of LysN in 150 mM ammonium bicarbonate The digestion was carried out using a 120 m/m ratio of enzyme to protein for 2 h at 55 C° The resulting peptides were purified using Pierce Grand Island NY USA C18 spin columns and the eluent was concentrated and resuspended in 5 acetonitrile for LC analysis An E coli lysate and proteins having one or more disulfide bonds were reduced using dithiothreitol DTT and acetylated using iodoacetamide prior to LysN digestion The peptides included in the tabulation of a/a + 1 ions are summarized in Supplemental Table 1All peptides were analyzed on a Thermo Scientific Orbitrap Fusion mass spectrometer San Jose CA USA modified by addition of 193 nm excimer laser as described previously 31 In brief an optical window was added to the manifold of the vacuum chamber at the back of the dual linear ion trap To enhance overlap of the laser beam with the ion cloud and thus improve UVPD efficiency the laser beam was focused using two planoconvex optical lenses The pulsing of the laser was timed to the normal CID activation period in the high pressure region of the linear ion trap and the laser was fired at 500 Hz which equates to 2 ms per pulse Detection in the Orbitrap mass analyzer is on a longer timescale requiring 100 ms or longer including ion transfer times For the extensive peptide data acquisition needed for the statistical analysis of hydrogen atom elimination in a + 1 ions liquid chromatography of LysNdigested peptides was performed using a Dionex nanoLC system coupled to the Orbitrap Fusion mass spectrometer Peptides were separated on a 24 cm C18 column packed inhouse and were eluted from the column using a 30 min gradient during which the solvent composition was varied from 15 acetonitrile to 60 acetonitrile in water with 01 formic acid Ions were generated by ESI via application of 1800 V to a solventvoltage tee and were transmitted to the linear ion trap for UVPD UVPD was performed using two pulses with 15 mJ delivered per pulse Precursor and product ion spectra were acquired using the Orbitrap mass analyzer a resolving power of 30 k was used for precursor ion spectra and a resolving power of 60 k was used for UVPD product ion spectra

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