Collision energies: Optimization strategies for bottom-up proteomics.

Mass-spectrometry coupled to liquid chromatography is an indispensable tool in the field of proteomics. In the last decades, more and more complex and diverse biochemical and biomedical questions have arisen. Problems to be solved involve protein identification, quantitative analysis, screening of low abundance modifications, handling matrix effect, and concentrations differing by orders of magnitude. This led the development of more tailored protocols and problem centered proteomics workflows, including advanced choice of experimental parameters. In the most widespread bottom-up approach, the choice of collision energy in tandem mass spectrometric experiments has outstanding role. This review presents the collision energy optimization strategies in the field of proteomics which can help fully exploit the potential of MS based proteomics techniques. A systematic collection of use case studies is then presented to serve as a starting point for related further scientific work. Finally, this article discusses the issue of comparing results from different studies or obtained on different instruments, and it gives some hints on methodology transfer between laboratories based on measurement of reference species.

Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides.

Identification and characterization of N-glycopeptides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results ("score"). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument. The resulting unprecedented amount of peptide-level information on such a large and diverse set of N-glycopeptides revealed that the peptide sequence heavily influences the energy for the highest score on top of an expected general linear trend with m/z. Search engine dependence may also be noteworthy. Based on the trends, we designed an experimental method and tested it on HeLa, blood plasma, and monoclonal antibody samples. As compared to the literature, these notably lower collision energies in our workflow led to 10-50% more identified N-glycopeptides, with higher scores. We recommend a simple approach based on a small set of reference N-glycopeptides easily accessible from glycoprotein standards to ease the precise determination of optimal methods on other instruments. Data sets can be accessed via the MassIVE repository (MSV000089657 and MSV000090218).

Tailoring to Search Engines: Bottom-Up Proteomics with Collision Energies Optimized for Identification Confidence.

Bottom-up proteomics relies on identification of peptides from tandem mass spectra, usually via matching against sequence databases. Confidence in a peptide-spectrum match can be characterized by a score value given by the database search engines, and it depends on the information content and the quality of the spectrum. The latter are influenced by experimental parameters, of which the collision energy is the most important one in the case of collision-induced dissociation. We examined how the identification score of the Byonic and Andromeda (MaxQuant) engines varies with collision energy for more than a thousand individual peptides from a HeLa tryptic digest on a QTof instrument. We thereby extended our earlier study on Mascot scores and corroborated its findings on the potential bimodal nature of this energy dependence. Optimal energies as a function of m/z show comparable linear trends for the three engines. On the basis of peptide-level results, we designed methods with one or two liquid chromatography-tandem mass spectrometry (LC-MS/MS) runs and various collision energy settings and assessed their practical performance in peptide and protein identification from the HeLa standard sample. A 10-40% gain in various measures, such as the number of identified proteins or sequence coverage, was obtained over the factory default settings. Best performing methods differ for the three engines, suggesting that the experimental parameters should be fine-tuned to the choice of the engine. We also recommend a simple approach and provide reference data to ease the transfer of the optimized methods to other mass spectrometers relevant for proteomics. We demonstrate the utility of this approach on an Orbitrap instrument. Data sets can be accessed via the MassIVE repository (MSV000086379).

Optimal Collision Energies and Bioinformatics Tools for Efficient Bottom-up Sequence Validation of Monoclonal Antibodies.

Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biopharmaceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and they often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of tandem mass spectroscopy (MS/MS) collision energies and automated combination of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits. We created a software (called "Serac") that consumes results of the Mascot database search engine and identifies the amino acids validated by bottom-up MS/MS experiments using the most rigorous, industrially acceptable definition of sequence coverage (we term this "confirmed sequence coverage"). The software can combine spectra at the level of amino acids or fragment ions to exploit complementarity, provides full transparency to justify validation, and reduces manual effort. With its help, we investigated collision energy dependence of confirmed sequence coverage of individual peptides and full proteins on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab). We found the energy dependence to be modest, but we demonstrated the benefit of using spectra taken at multiple energies. We describe a workflow based on 2-3 LC-MS/MS runs, carefully selected collision energies, and a fragment ion level combination, which yields ∼85% confirmed sequence coverage, 25%-30% above that from a basic proteomics protocol. Further increase can mainly be expected from alternative digestion enzymes or fragmentation techniques, which can be seamlessly integrated to the processing, thereby allowing effortless validation of full sequences.

Selection of Collision Energies in Proteomics Mass Spectrometry Experiments for Best Peptide Identification: Study of Mascot Score Energy Dependence Reveals Double Optimum.

Collision energy is a key parameter determining the information content of beam-type collision induced dissociation tandem mass spectrometry (MS/MS) spectra, and its optimal choice largely affects successful peptide and protein identification in MS-based proteomics. For an MS/MS spectrum, quality of peptide match based on sequence database search, often characterized in terms of a single score, is a complex function of spectrum characteristics, and its collision energy dependence has remained largely unexplored. We carried out electrospray ionization-quadrupole-time of flight (ESI-Q-TOF)-MS/MS measurements on 2807 peptides from tryptic digests of HeLa and E. coli at 21 different collision energies. Agglomerative clustering of the resulting Mascot score versus energy curves revealed that only few of them display a single, well-defined maximum; rather, they feature either a broad plateau or two clear peaks. Nonlinear least-squares fitting of one or two Gaussian functions allowed the characteristic energies to be determined. We found that the double peaks and the plateaus in Mascot score can be associated with the different energy dependence of b- and y-type fragment ion intensities. We determined that the energies for optimum Mascot scores follow separate linear trends for the unimodal and bimodal cases with rather large residual variance even after differences in proton mobility are taken into account. This leaves room for experiment optimization and points to the possible influence of further factors beyond m/ z.

Stereocontrol in Diphenylprolinol Silyl Ether Catalyzed Michael Additions: Steric Shielding or Curtin-Hammett Scenario?

The enantioselectivity of amine-catalyzed reactions of aldehydes with electrophiles is often explained by simple steric arguments emphasizing the role of the bulky group of the catalyst that prevents the approach of the electrophile from the more hindered side. This standard steric shielding model has recently been challenged by the discovery of stable downstream intermediates, which appear to be involved in the rate-determining step of the catalytic cycle. The alternative model, referred to as the Curtin-Hammett scenario of stereocontrol, assumes that the enantioselectivity is related to the stability and reactivity of downstream intermediates. In our present computational study, we examine the two key processes of the catalytic Michael reaction between propanal and ß-nitrostyrene that are relevant to the proposed stereoselectivity models, namely the C-C bond formation and the protonation steps. The free energy profiles obtained for the pathways leading to the enantiomeric products suggest that the rate- and stereodetermining steps are not identical as implied by the previous models. The stereoselectivity can be primarily controlled by C-C bond formation even though the reaction rate is dictated by the protonation step. This kinetic scheme is consistent with all observations of experimental mechanistic studies including those of mass spectrometric back reaction screening experiments, which reveal a mismatch between the stereoselectivity of the back and the forward reactions.

Composite aromatic boxes for enzymatic transformations of quaternary ammonium substrates.

Cation-π interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors.

Can We Boost N-Glycopeptide Identification Confidence? Smart Collision Energy Choice Taking into Account Structure and Search Engine.

High confidence and reproducibility are still challenges in bottom-up mass spectrometric N-glycopeptide identification. The collision energy used in the MS/MS measurements and the database search engine used to identify the species are perhaps the two most decisive factors. We investigated how the structural features of N-glycopeptides and the choice of the search engine influence the optimal collision energy, delivering the highest identification confidence. We carried out LC-MS/MS measurements using a series of collision energies on a large set of N-glycopeptides with both the glycan and peptide part varied and studied the behavior of Byonic, pGlyco, and GlycoQuest scores. We found that search engines show a range of behavior between peptide-centric and glycan-centric, which manifests itself already in the dependence of optimal collision energy on m/z. Using classical statistical and machine learning methods, we revealed that peptide hydrophobicity, glycan and peptide masses, and the number of mobile protons also have significant and search-engine-dependent influence, as opposed to a series of other parameters we probed. We envisioned an MS/MS workflow making a smart collision energy choice based on online available features such as the hydrophobicity (described by retention time) and glycan mass (potentially available from a scout MS/MS). Our assessment suggests that this workflow can lead to a significant gain (up to 100%) in the identification confidence, particularly for low-scoring hits close to the filtering limit, which has the potential to enhance reproducibility of N-glycopeptide analyses. Data are available via MassIVE (MSV000093110).

Optimum collision energies for proteomics: The impact of ion mobility separation.

Ion mobility spectrometry (IMS) is a widespread separation technique used in various research fields. It can be coupled to liquid chromatography-mass spectrometry (LC-MS/MS) methods providing an additional separation dimension. During IMS, ions are subjected to multiple collisions with buffer gas, which may cause significant ion heating. The present project addresses this phenomenon from the bottom-up proteomics point of view. We performed LC-MS/MS measurements on a cyclic ion mobility mass spectrometer with varied collision energy (CE) settings both with and without IMS. We investigated the CE dependence of identification score, using Byonic search engine, for more than 1000 tryptic peptides from HeLa digest standard. We determined the optimal CE values-giving the highest identification score-for both setups (i.e., with and without IMS). Results show that lower CE is advantageous when IMS separation is applied, by 6.3 V on average. This value belongs to the one-cycle separation configuration, and multiple cycles may supposedly have even larger impact. The effect of IMS is also reflected in the trends of optimal CE values versus m/z functions. The parameters suggested by the manufacturer were found to be almost optimal for the setup without IMS; on the other hand, they are obviously too high with IMS. Practical consideration on setting up a mass spectrometric platform hyphenated to IMS is also presented. Furthermore, the two CID (collision induced dissociation) fragmentation cells of the instrument-located before and after the IMS cell-were also compared, and we found that CE adjustment is needed when the trap cell is used for activation instead of the transfer cell. Data have been deposited in the MassIVE repository (MSV000090944).

Identification and interconversion of diastereomeric oligo-Tröger bases probed by ion mobility mass spectrometry.

Oligo-Tröger bases are auspicious scaffolds of molecular engineering, which motivates studies on the mechanism of their interconversion and on the facile determination of the relative configuration of their diastereoisomers. Protonated, sodiated, and argentated species of those compounds were therefore studied via ion-mobility mass spectrometry (IM-MS), allowing differentiation on the basis of the shapes of the ions. First, the isomerization was confirmed to be acid-catalyzed as it takes place readily in the case of protonated Tröger bases, whereas the metallated bases are configurationally stable. Second, the corrected arrival times of the various isomers of the cationized bases were found to show distinct differences in IM-MS, and their excellent correlation with the cross sections obtained from quantum chemical calculations paves the way toward the easy identification of diastereoisomers.

Stepwise Collision Energy-Resolved Tandem Mass Spectrometric Experiments for the Improved Identification of Citrullinated Peptides.

The use of tandem mass spectrometry (MS/MS) is a fundamental prerequisite of reliable protein identification and quantification in mass-spectrometry-based proteomics. In bottom-up and middle-down proteomics, proteins are identified by the characteristic fragments of their constituting peptides. Post-translational modifications (PTMs) often further complicate proteome analyses. Citrullination is an increasingly studied PTM converting arginines to citrullines (Cit, X) and is implicated in several autoimmune and neurological diseases as well as different types of cancer. Confirmation of citrullination is known to be very challenging since it results in the same molecular mass change as Asn/Gln deamidation. In this study, we explore which MS/MS characteristics can be used for the reliable identification of citrullination. We synthesized several peptides incorporating Cit residues that model enzymatic cleavages of different proteins with verified or putative citrullination. Collision-induced dissociation was used to investigate the energy dependence of Byonic and Mascot scores and confirmed sequence coverage (CSC) along with the neutral loss of HNCO characteristic to citrulline side chains. We found that although the recommended values (19-45 V) for ramped collision energy settings cover the optimal Mascot, Byonic, or %CSC scores effectively, the diagnostic HNCO loss from precursors and fragments may reach their maximum intensities at lower and higher collision energies, respectively. Therefore, we suggest broadening the ramp range to ∼5-60 V to obtain more favorable identification rates for citrullinated peptides. We also found that Byonic was more successful in correctly identifying citrullinated peptides with deamidated residues than Mascot.

Identification of Intrinsically Disordered Proteins and Regions in a Non-Model Insect Species Ostrinia nubilalis (Hbn.).

Research in previous decades has shown that intrinsically disordered proteins (IDPs) and regions in proteins (IDRs) are as ubiquitous as highly ordered proteins. Despite this, research on IDPs and IDRs still has many gaps left to fill. Here, we present an approach that combines wet lab methods with bioinformatics tools to identify and analyze intrinsically disordered proteins in a non-model insect species that is cold-hardy. Due to their known resilience to the effects of extreme temperatures, these proteins likely play important roles in this insect's adaptive mechanisms to sub-zero temperatures. The approach involves IDP enrichment by sample heating and double-digestion of proteins, followed by peptide and protein identification. Next, proteins are bioinformatically analyzed for disorder content, presence of long disordered regions, amino acid composition, and processes they are involved in. Finally, IDP detection is validated with an in-house 2D PAGE. In total, 608 unique proteins were identified, with 39 being mostly disordered, 100 partially disordered, 95 nearly ordered, and 374 ordered. One-third contain at least one long disordered segment. Functional information was available for only 90 proteins with intrinsic disorders out of 312 characterized proteins. Around half of the 90 proteins are cytoskeletal elements or involved in translational processes.

On the mechanism of the copper-mediated C-S bond formation in the intramolecular disproportionation of imine disulfides.

The mechanism of the copper-mediated disproportionation of aromatic imine disulfides to benzothiazoles in the gas phase is investigated by experimental and theoretical methods. Application of infrared multiphoton dissociation and hydrogen/deuterium exchange experiments combined with density functional theory (DFT) calculations of the relevant molecular structures and the associated infrared spectra allows the identification of the observed ionic intermediates. The theoretical investigation of the possible reaction pathways supported by collision-induced dissociation experiments provides a consistent mechanistic picture of the reaction catalyzed by a single copper(I) ion. Activation of the substrate proceeds via homolytic sulfur-sulfur bond cleavage, yielding metal complexes in the formal +3 oxidation state; carbon-sulfur coupling and hydrogen-atom transfer complete the transformation to the products. Exploratory studies demonstrate that in the gas phase, the disproportionation of the imine disulfide can also be mediated by other metal ions via different either homo- or heterolytic mechanisms without involving high-valent intermediates.

Anion binding by bambus[6]uril probed in the gas phase and in solution.

Electrospray ionization mass spectrometry (ESI-MS) is used to probe the binding of small anions to the macrocycle of bambus[6]uril. For the halide ions, the experimental patterns suggest F(-) < Cl(-) < Br(-) < I(-), which is consistent with the order of anion binding found in the condensed phase. Parallel equilibrium studies in the condensed phase establish the association constants of halide anions and bambus[6]uril in mixed solvents. A detailed analysis of the mass spectrometric data is used to shed light on the correlations between the binding constants in the condensed phase and the ion abundances observed using ESI-MS. From the analysis it becomes apparent that ESI-MS can indeed represent the situation in solution to some extent, but the sampling in the gas-phase experiment is not 1:1 compared to that in solution.

Microhydration of the magnesium(II) acetate cation in the gas phase.

Electrospray ionization of aqueous solutions of magnesium(II) acetate leads to microhydrated magnesium acetate cations of the type [(CH(3)COO)(2m-1)Mg(m)(H(2)O)(n)](+) with m = 1-4 and n = 0-4, which are characterized by mass spectrometry and, for the cluster with three water molecules, also by infrared multiphoton dissociation spectroscopy. Density functional theory is used to determine the energies of microhydration for the mononuclear species [(CH(3)COO)Mg(H(2)O)(n)](+) with n = 0-6 and the associated changes in molecular structure. While bidentate coordination of the acetato ligand is generally preferred, at higher values of n, a switch to a monodentate coordination becomes energetically competitive.

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

Quadrupole time-of-flight (QTof) collision-induced dissociation (CID) and Orbitrap higher-energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry-based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q-Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap-specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434).

Influence of Post-Translational Modifications on Protein Identification in Database Searches.

Comprehensive analysis of post-translation modifications (PTMs) is an important mission of proteomics. However, the consideration of PTMs increases the search space and may therefore impair the efficiency of protein identification. Using thousands of proteomic searches, we investigated the practical aspects of considering multiple PTMs in Byonic searches for the maximization of protein and peptide hits. The inclusion of all PTMs, which occur with at least 2% frequency in the sample, has an advantageous effect on protein and peptide identification. A linear relationship was established between the number of considered PTMs and the number of reliably identified peptides and proteins. Even though they handle multiple modifications less efficiently, the results of MASCOT (using the Percolator function) and Andromeda (the search engine included in MaxQuant) became comparable to those of Byonic, in the case of a few PTMs.

Binding energies and isomerization in metallocene ions from threshold photoelectron photoion coincidence spectroscopy.

Metallocene ions (Cp(2)M(+), M = Cr, Co, Ni) were studied by threshold photoelectron photoion coincidence spectroscopy (TPEPICO) to investigate the mechanism, energetics, and kinetics of the ionic dissociation processes. The examined energy-selected Cp(2)M(+) ions fragment by losing the neutral cyclopentadienyl ligand. In addition, CH and C(2)H(2) losses appear as minor channels, while the cobaltocene ion also loses an H atom. A possible isomerization pathway has also been observed for Cp(2)Ni(+), yielding a complex with pentafulvalene (C(10)H(8)) with a loss of H(2). In order to determine the 0 K appearance energies for the CpM(+) fragment ions, the asymmetric time-of-flight peak shapes and the breakdown diagrams of the energy-selected metallocene ions were modeled by both the rigid activated complex (RAC) Rice-Ramsperger-Kassel-Marcus (RRKM) theory and the simplified statistical adiabatic channel model (SSACM). The following appearance energies were obtained with SSACM, which is more reliable for loose transition states: 10.57 ± 0.14, 11.01 ± 0.13, and 10.18 ± 0.13 eV for M = Cr, Co, and Ni, respectively. These values combined with the corresponding adiabatic ionization energies yield M-Cp bond dissociation energies in Cp(2)M(+) ions of 5.04 ± 0.16, 5.77 ± 0.15, and 3.96 ± 0.15 eV. Density functional calculations at the B3LYP/6-311G(d,p) level of theory were used to determine the structures of these complexes and to provide parameters necessary for the analysis of the experimental data. The trends in the M-Cp bond energies can be related to the electronic structures of the metallocene ions based on a simple molecular orbital picture.

Mechanistic insights into a copper-disulfide interaction in oxidation of imines by disulfides.

The concept of using disulfides as an oxidant for Cu(i) is introduced as part of a Cu-catalyzed process leading to the formation of benzothiazole from an iminodisulfide under an inert atmosphere.