Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions

For decades, formaldehyde has been routinely used to cross-link proteins in cells, tissue, and in some instances, even entire organisms. Due to its small size, formaldehyde can readily permeate cell walls and membranes, resulting in efficient cross-linking, i.e. the formation of covalent bonds between proteins, DNA, and other reactive molecules. Indeed, formaldehyde cross-linking is an instrumental component of many mainstream analytical/cell biology techniques including chromatin immunoprecipitation (ChIP) of protein-DNA complexes found in nuclei; immunohistological analysis of protein expression and localization within cells, tissues, and organs; and mass spectrometry (MS)-compatible silver-staining methodologies used to visualize low abundance proteins in polyacrylamide gels. However, despite its exquisite suitability for use in the analysis of protein environments within cells, formaldehyde has yet to be commonly employed in the directed analysis of protein-protein interactions and cellular networks. The general purpose of this article is to discuss recent advancements in the use of formaldehyde cross-linking in combination with MS-based methodologies. Key advantages and limitations to the use of formaldehyde over other cross-linkers and technologies currently used to study protein-protein interactions are highlighted, and formaldehyde-based experimental approaches that are proving very promising in their ability to accurately and efficiently identify novel protein-protein and multiprotein interaction complexes are presented.     

Dissociation behavior of a bifunctional tempo‐active ester reagent for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) mass spectrometry

We have synthesized a homobifunctional active ester cross‐linking reagent containing a TEMPO (2,2,6,6‐tetramethylpiperidine‐1‐oxy) moiety connected to a benzyl group (Bz), termed TEMPO‐Bz‐linker. The aim for designing this novel cross‐linker was to facilitate MS analysis of cross‐linked products by free radical initiated peptide sequencing (FRIPS). The TEMPO‐Bz‐linker was reacted with all 20 proteinogenic amino acids as well as with model peptides to gain detailed insights into its fragmentation mechanism upon collision activation. The final goal of this proof‐of‐principle study was to evaluate the potential of the TEMPO‐Bz‐linker for chemical cross‐linking studies to derive 3D‐structure information of proteins. Our studies were motivated by the well documented instability of the central NO―C bond of TEMPO‐Bz reagents upon collision activation. The fragmentation of this specific bond was investigated in respect to charge states and amino acid composition of a large set of precursor ions resulting in the identification of two distinct fragmentation pathways. Molecular ions with highly basic residues are able to keep the charge carriers located, i.e. protons or sodium cations, and consequently decompose via a homolytic cleavage of the NO―C bond of the TEMPO‐Bz‐linker. This leads to the formation of complementary open‐shell peptide radical cations, while precursor ions that are protonated at the TEMPO‐Bz‐linker itself exhibit a charge‐driven formation of even‐electron product ions upon collision activation. MS³ product ion experiments provided amino acid sequence information and allowed determining the cross‐linking site. Our study fully characterizes the CID behavior of the TEMPO‐Bz‐linker and demonstrates its potential, but also its limitations for chemical cross‐linking applications utilizing the special features of open‐shell peptide ions on the basis of selective tandem MS analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes

Chemical cross-linking of proteins, an established method in protein chemistry, has gained renewed interest in combination with mass spectrometric analysis of the reaction products for elucidating low-resolution three-dimensional protein structures and interacting sequences in protein complexes. The identification of the large number of cross-linking sites from the complex mixtures generated by chemical cross-linking, however, remains a challenging task. This review describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the strategies employing intra- or intermolecular chemical cross-linking and mass spectrometry. The various approaches described in the literature to facilitate detection of cross-linking products and also computer software for data analysis are reviewed. Cross-linking techniques combined with mass spectrometry and bioinformatic methods have the potential to provide the basis for an efficient structural characterization of proteins and protein complexes.     

Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry

An analytical strategy for the analysis of antigen epitopes by chemical cross-linking and mass spectrometry is demonstrated. The information of antigen peptides involved in the binding to an antibody can be obtained by monitoring the antigen peptides modified by a partially hydrolyzed cross-linker in the absence and in the presence of an antibody. This approach was shown to be efficient for characterization of the epitope on bovine prion protein bPrP(25-241) specifically recognized by a monoclonal antibody, 3E7 (mAb3E7), with only a small amount of sample (200 picomoles) needed. After cross-linking of the specific immuno complex, a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer equipped with an ion conversion dynode (ICD) high-mass detector was used to optimize the amount of cross-linked complex formed at 202 kDa before proteolytic digestion. To identify the cross-linked peptides after proteolysis without ambiguity, isotope-labeled cross-linkers, disuccinimidyl suberate (DSS-d0/d12) and disuccinimidyl glutarate (DSG-d0/d6), together with high-resolution Fourier transform ion-cyclotron resonance mass spectrometry (FTICR-MS) were used. As a result, a complete fading of the peak intensities corresponding to the peptides representing the epitope was observed when bPrP/mAb3E7 complexes were formed.     

"Mass defect" tags for biomolecular mass spectrometry

We present a new class of "mass defect" tags with utility in biomolecular mass spectrometry. These tags, incorporating element(s) with atomic numbers between 17 (Cl) and 77 (Ir), have a substantially different nuclear binding energy (mass defect) from the elements common to biomolecules. This mass defect yields a readily resolvable mass difference between tagged and untagged species in high-resolution mass spectrometers. We present the use of a subset of these tags in a new protein sequencing application. This sequencing technique has advantages over existing mass spectral protein identification methodologies: intact proteins are quickly sequenced and unambiguously identified using only an inexpensive, robust mass spectrometer. We discuss the potential broader utility of these tags for the sequencing of other biomolecules, differential display applications and combinatorial methods.     

Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry

We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.     

Fragmentation features of intermolecular cross‐linked peptides using N‐hydroxy‐ succinimide esters by MALDI‐ and ESI‐MS/MS for use in structural proteomics

The use of mass spectrometry coupled with chemical cross‐linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross‐linked peptides. The interpretation of the MS/MS data generated in cross‐linking experiments using N‐hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross‐linked peptides occur when two different peptides are connected by the cross‐linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross‐linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross‐linked peptides present in cross‐linking experiments and be further implemented in search engine's algorithms. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification of leucine-enkephalin radical oxidation products by liquid chromatography tandem mass spectrometry

The oxidation of the peptide leucine-enkephalin (YGGFL) induced by the hydroxyl radical (HO*), formed under Fenton-like conditions [Cu (II)/H(2)O(2)], was studied and monitored by LC-MS. The oxidation products identified included products resultant from (a) the insertion of oxygen atoms (1-5), (b) peptide backbone cleavage (short-chain products formed by diamide pathway) and (c) radical-radical crosslinking reactions. In order to identify the modified residues, LC-MS/MS spectra were obtained. The insertion of oxygen atoms into the peptide originated hydroxide, di-hydroxide and/or hydroperoxide derivatives. In addition it was found that the aromatic amino acids are most susceptible to being hydroxylated, while the aliphatic amino acids are more prone to forming hydroperoxides. Oxidation products with double bonds were also identified. The short chain products resulted from the alpha-carbon radical of terminal amino acids (Tyr and Leu). Products resulting from cross-linking reactions between intact carbon-centered peptide radical (with and without one HO group) and a side chain radical (*C(7)H(7)O) were identified. It was found that, although all amino acids residues of the peptide undergo modifications, the N-terminal seems to be prone to oxidative modifications under these conditions.     

Metabolite identification of the antimalarial naphthoquine using liquid chromatography–tandem high‐resolution mass spectrometry in combination with multiple data‐mining tools

Naphthoquine (NQ) is one of important partner drugs of artemisinin‐based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2–4 h) and has a long elimination half‐life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMSⁿ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid‐phase extraction. For data processing, multiple data‐mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1–M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N‐oxidation. This study demonstrates that LC‐HRMSⁿ in combination with multiple data‐mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.     

Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.     

Elucidating the higher‐order structure of biopolymers by structural probing and mass spectrometry: MS3D

Chemical probing represents a very versatile alternative for studying the structure and dynamics of substrates that are intractable by established high‐resolution techniques. The implementation of MS‐based strategies for the characterization of probing products has not only extended the range of applicability to virtually all types of biopolymers but has also paved the way for the introduction of new reagents that would not have been viable with traditional analytical platforms. As the availability of probing data is steadily increasing on the wings of the development of dedicated interpretation aids, powerful computational approaches have been explored to enable the effective utilization of such information to generate valid molecular models. This combination of factors has contributed to making the possibility of obtaining actual 3D structures by MS‐based technologies (MS3D) a reality. Although approaches for achieving structure determination of unknown targets or assessing the dynamics of known structures may share similar reagents and development trajectories, they clearly involve distinctive experimental strategies, analytical concerns and interpretation paradigms. This Perspective offers a commentary on methods aimed at obtaining distance constraints for the modeling of full‐fledged structures while highlighting common elements, salient distinctions and complementary capabilities exhibited by methods used in dynamics studies. We discuss critical factors to be addressed for completing effective structural determinations and expose possible pitfalls of chemical methods. We survey programs developed for facilitating the interpretation of experimental data and discuss possible computational strategies for translating sparse spatial constraints into all‐atom models. Examples are provided to illustrate how the concerted application of very diverse probing techniques can lead to the solution of actual biological systems. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass defect filter technique and its applications to drug metabolite identification by high‐resolution mass spectrometry

Identification of drug metabolites by liquid chromatography/mass spectrometry (LC/MS) involves metabolite detection in biological matrixes and structural characterization based on product ion spectra. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of metabolites using triple‐quadrupole and ion trap mass spectrometers. Recently, a novel mass defect filter (MDF) technique has been developed, which enables high‐resolution mass spectrometers to be utilized for detecting both predicted and unexpected drug metabolites based on narrow, well‐defined mass defect ranges for these metabolites. This is a new approach that is completely different from, but complementary to, traditional molecular mass‐ or MS/MS fragmentation‐based LC/MS approaches. This article reviews the mass defect patterns of various classes of drug metabolites and the basic principles of the MDF approach. Examples are given on the applications of the MDF technique to the detection of stable and chemically reactive metabolites in vitro and in vivo. Advantages, limitations, and future applications are also discussed on MDF and its combinations with other data mining techniques for the detection and identification of drug metabolites. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of radiation-induced cross-linking between thymine and tryptophan by electrospray ionization-mass spectrometry

Upon exposure to ionizing radiation, DNA undergoes a variety of modifications including the production of a covalent bond between the nucleobase thymine and aromatic amino acids. In this work, electrospray ionization-mass spectrometry(ESI-MS) was used to identify the gamma radiation-induced covalent cross-linking of model peptides (sequence YPPW and pYPPW) with the nucleobase thymine. Tandem electrospray ionization-mass spectrometry (ESI-MSn) was employed to investigate the cross-linking sites. The results showed that irrespective of whether tyrosine was phosphorylated or not, the nucleobase thymine was cross-linked with the tryptophan residue. Possible cross-linking mechanisms are proposed by investigating the related mass peaks.     

Reactivity of Tyr–Leu and Leu–Tyr dipeptides: identification of oxidation products by liquid chromatography–tandem mass spectrometry

The exposure of peptides and proteins to reactive hydroxyl radicals results in covalent modifications of amino acid side‐chains and protein backbone. In this study we have investigated the oxidation the isomeric peptides tyrosine–leucine (YL) and leucine–tyrosine (LY), by the hydroxyl radical formed under Fenton reaction (Fe²⁺/H₂O₂). Through mass spectrometry (MS), high‐performance liquid chromatography (HPLC‐MS) and electrospray tandem mass spectrometry (HPLC‐MSⁿ) measurements, we have identified and characterized the oxidation products of these two dipeptides. This approach allowed observing and identifying a wide variety of oxidation products, including isomeric forms of the oxidized dipeptides. We detected oxidation products with 1, 2, 3 and 4 oxygen atoms for both peptides; however, oxidation products with 5 oxygen atoms were only present in LY. LY dipeptide oxidation leads to more isomers with 1 and 2 oxygen atoms than YL (3 vs 5 and 4 vs 5, respectively). Formation of the peroxy group occurred preferentially in the C‐terminal residue. We have also detected oxidation products with double bonds or keto groups, dimers (YL–YL and LY–LY) and other products as a result of cross‐linking. Both amino acids in the dipeptides were oxidized although the peptides showed different oxidation products. Also, amino acid residues have shown different oxidation products depending on the relative position on the dipeptide. Results suggest that amino acids in the C‐terminal position are more prone to oxidation. Copyright © 2009 John Wiley & Sons, Ltd.     

Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry

The combination of gel-based two-dimensional protein separations with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the workhorse for the large-scale analyses of proteomes. Such high-throughput proteomic approaches require automation of all post-separation steps and the in-gel digest of proteins especially is often the bottleneck in the protein identification workflow. With the objective of reaching the same high performance of manual low-throughput in-gel digest procedures, we have developed a novel stack-type digestion device and implemented it into a commercially available robotic liquid handling system. This modified system is capable of performing in-gel digest, extraction of proteolytic peptides, and subsequent sample preparation for MALDI-MS without any manual intervention, but with a performance at least identical to manual procedures as indicated on the basis of the sequence coverage obtained by peptide mass fingerprinting. For further refinement of the automated protein identification workflow, we have also developed a motor-operated matrix application device to reproducibly obtain homogenous matrix preparation of high quality. This matrix preparation was found to be suitable for the automated acquisition of both peptide mass fingerprint and fragment ion spectra from the same sample spot, a prerequisite for high confidence protein identifications on the basis of peptide mass and sequence information. Due to the implementation of the stack-type digestion device and the motor-operated matrix application device, the entire platform works in a reliable, cost-effective, and sensitive manner, yielding high confidence protein identifications even for samples in the concentration range of as low as 100 fmol protein per gel plug.      

Rapid identification of pesticides in human oral fluid for emergency management by thermal desorption electrospray ionization/mass spectrometry

Self‐poisoning with pesticides accounts for approximately one‐third of all suicides worldwide. To expedite rescue in the emergency department, it is essential to develop a point‐of‐care analytical method for rapid identification of ingested pesticides. In this study, five of the most common pesticides ingested by self‐poisoning patients in Taiwan were analyzed from oral fluid samples. Pesticide‐oral fluid mixtures were applied on a cotton swab and then transferred into methanol. A metallic probe was used to sample the methanol solution for subsequent thermal desorption‐electrospray ionization mass spectrometry analysis. Altogether, pesticide sampling, transfer, desorption, ionization, and detection took less than 1 min. The reproducibility of this method (n = 6) was shown in the observed low‐relative standard deviation (<7%) in the detection of pesticide in oral fluid. The detection limits of the pesticides in oral fluid obtained from four human subjects by thermal desorption‐electrospray ionization mass spectrometry were between 1–10 ppb with relative standard deviation 10.7%. Moreover, in this study, linear responses of five pesticides in oral fluid with concentrations between 1 ppb–1 ppm (R2 between 0.9938 and 0.9988) were observed. As the whole analytical process is extremely short, this technique allows for early non‐invasive point‐of‐care identification of pesticides in the oral fluid of self‐poisoning patients in the emergency room, providing important toxicological information for decision‐making during critical resuscitation. Copyright © 2016 John Wiley & Sons, Ltd.     

液相色谱-高分辨质谱联用技术在食品欺诈检测鉴别中的应用

针对我国现阶段较为突出的违法使用农兽药、滥用食品添加剂、非法添加非食用物质和掺杂使假等食品欺诈问题,综述了近5年来液相色谱-高分辨质谱联用技术在该领域的应用情况。重点介绍了高分辨质谱,主要是飞行时间质谱和静电场轨道阱质谱技术的定向筛查和非定向筛查功能在食品欺诈检测鉴别中的应用,以期为解决该类食品安全问题提供有益的参考。     

Structurally diagnostic ion/molecule reactions: class and functional-group identification by mass spectrometry

This article discusses the application of gas-phase ion/molecule reactions for fine structural elucidation in mass spectrometry. This approach is illustrated via a representative collection of class- and functional group-selective reactions, a few of historical relevance as well as by more recent and instructive examples, and their applications. The focus is on reactions performed under well-controlled conditions of sequential mass spectrometry, discussing key mechanistic details and potential applications. Recent and innovative strategies that allow these reactions to be performed under ambient conditions, making this fast, selective and sensitive approach for structural investigation much more generally applicable, are also discussed.