Ambient ionisation mass spectrometry for in situ analysis of intact proteins

Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed.     

Identification of Regions in Apomyoglobin that Form Intermolecular Interactions in Amyloid Aggregates Using High-Performance Mass Spectrometry

The formation of amyloid aggregates in human organs and tissues causes the development of incurable diseases. However, experimental studies of the mechanism of amyloid formation by proteins and the structural characteristics of amyloids are complicated because of the heterogeneity and high molecular weight of the aggregates. We used limited proteolysis and mass spectrometry for the identification of regions in the apomyoglobin polypeptide chain, which give rise to intermolecular interactions in amyloid structures. Tandem mass spectroscopy enabled the identification of regions in the myoglobin polypeptide chain, which form the core of amyloid structures. It was shown that the main structural elements for the formation of the core of amyloid fibrils in myoglobin were regions from 60 through 90 and from 97 through 124 amino acid residues. These regions coincide well with those theoretically predicted. This approach yielded important data on the structure of protein molecules in aggregates and on conformational rearrangements of apomyoglobin upon amyloid formation.     

LC-mass spectrometry analysis of N- and C-terminal boundary sequences of polypeptide fragments by limited proteolysis

Limited proteolysis is an important and widely used method for analyzing the tertiary structure and determining the domain boundaries of proteins. Here we describe a novel method for determining the N- and C-terminal boundary amino acid sequences of products derived from limited proteolysis using semi-specific and/or non-specific enzymes, with mass spectrometry as the only analytical tool. The core of this method is founded on the recognition that cleavage of proteins with non-specific proteases is not random, but patterned. Based on this recognition, we have the ability to determine the sequence of each proteolytic fragment by extracting a common association between data sets containing multiple potential sequences derived from two or more different mass spectral molecular weight measurements. Proteolytic product sequences derived from specific and non-specific enzymes can be accurately determined without resorting to the conventional time-consuming and laborious methods of SDS-PAGE and N-terminal sequencing analysis. Because of the sensitivity of mass spectrometry, multiple transient proteolysis intermediates can also be identified and analyzed by this method, which allows the ability to monitor the progression of proteolysis and thereby gain insight into protein structures.     

Mass spectrometry in the characterization of cereal seed proteins

In less then a decade, applications of matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) mass spectrometry to the investigation of prolamins have rapidly evolved from measurements of the molecular mass of isolated proteins to a proteomic approach attempting to characterise the complete protein pattern in the seed. Mass spectrometry is currently making significant contributions to the understanding of the composition and structure of the gluten proteins and, in turn, to the elucidation of structure-function relationships. Results obtained using mass spectrometry, including determination of the molecular masses of prolamins, direct verification of gene-derived sequences, determination of the number of cysteine residues and localisation of disulphide bonds, investigation of the gluten toxicity for celiac patients, qualitative and quantitative determination of gliadins in food and determination of the protein pattern and its modification during seed maturation by proteomic approaches, are summarised here, to illustrate current trends and individuate possible future perspectives.     

Preliminary mechanistic information on disulfide-bond formation and the role of hydrogen bonds by nanoelectrospray mass spectrometry

The formation of disulfide-bonds is vital for the proper folding of most secreted proteins and the stabilization of the final protein structure, including many of medical importance. The determination of disulfide-bonds is an important aspect of gaining a comprehensive understanding of the chemical structure of a protein. A long-term goal of ours is to examine the mechanism of disulfide-bond formation in aqueous solution and the potential role hydrogen bonds play in this process. Here, we report preliminary results from a method that utilizes the oxidizing power of iodine to generate disulfide bonds from synthesized model compounds, which is followed by nanoelectrospray ionization (nanoESI)- mass spectrometry (MS). By continuously monitoring the reaction mixture during disulfide formation, this nanoESI approach provides insight on the sequence of intermediate species formed, and how hydrogen-bonding donor/acceptor pairs may promote disulfide bond formation.     

Enhanced specificity of bacterial spore identification by oxidation and mass spectrometry

Addition of an oxidizing agent (e.g., hydrogen peroxide) to intact spores selectively and completely oxidizes Met-containing biomarker proteins by formation of Met sulfoxides. This reaction increases the masses of the biomarker proteins observed in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of Bacillus spores by Deltam = (16 x n) Da, where n is the number of Met residues in the sequence of each individual protein. The procedure is very rapid, and can be performed in situ (i.e., on the MALDI target). It confirms the identity of individual biomarkers by comparing the number of Met amino acids from the experimentally determined mass shifts with predictions for n from the tentative amino acid sequence for each protein. In turn, accurate determination of n for several biomarkers allows rapid validation of the initial spore identification by MALDI-MS.     

Accelerated proteolysis in alternating electric fields for peptide mapping

Sinusoidal alternating voltages (typically 5 V) were employed to enhance the efficiency of proteolysis for peptide mapping in this work. Protein solutions containing trypsin were allowed to digest with the assistance of alternating electric fields (AEFs) between a pair of platinum wire electrodes in Eppendorf tubes. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of several standard proteins. It was demonstrated that AEFs significantly accelerated in-solution proteolysis and the digestion time was substantially reduced to 5 min. The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with sequence coverages that were comparable to those obtained by using conventional 12-h in-solution proteolysis. The suitability of AEF-assisted proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). The present proteolysis strategy is simple and efficient and will find a wide range of applications in protein identification.     

Rapid 3-dimensional shape determination of globular proteins by mobility capillary electrophoresis and native mass spectrometry

Established high-throughput proteomics methods provide limited information on the stereostructures of proteins. Traditional technologies for protein structure determination typically require laborious steps and cannot be performed in a high-throughput fashion. Here, we report a new medium throughput method by combining mobility capillary electrophoresis (MCE) and native mass spectrometry (MS) for the 3-dimensional (3D) shape determination of globular proteins in the liquid phase, which provides both the geometric structure and molecular mass information of proteins. A theory was established to correlate the ion hydrodynamic radius and charge state distribution in the native mass spectrum with protein geometrical parameters, through which a low-resolution structure (shape) of the protein could be determined. Our test data of 11 different globular proteins showed that this approach allows us to determine the shapes of individual proteins, protein complexes and proteins in a mixture, and to monitor protein conformational changes. Besides providing complementary protein structure information and having mixture analysis capability, this MCE and native MS based method is fast in speed and low in sample consumption, making it potentially applicable in top–down proteomics and structural biology for intact globular protein or protein complex analysis.

Using native mass spectrometry and mobility capillary electrophoresis, the ellipsoid dimensions of globular proteins or protein complexes could be measured efficiently.     

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights. Larger proteolytic peptides can be directly identified by the combination of measured mass, enzyme specificity, and protein database searching. Sucrose density gradient centrifugation revealed that the majority of the 70S ribosome dissociates into intact 30S and 50S subunits after 120 min of limited proteolysis. Thus, examination of ribosome populations within the first 30 to 60 min of incubation provides insight into 70S structural features. Results from E. coli and T. thermophilus revealed that a significantly larger fraction of 50S ribosomal proteins have similar limited proteolysis behavior than the 30S ribosomal proteins of these two organisms. The data obtained by this approach correlate with information available from the high-resolution crystal structures of both organisms. This new approach will be applicable to investigations of other large ribonucleoprotein complexes, is readily extendable to ribosomes from other organisms, and can facilitate additional structural studies on ribosome assembly intermediates.     

A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through "kinetic optimization" of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip.     

Electrospray-assisted modification of proteins: a radical probe of protein structure

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.     

Mass spectrometry-based structural proteomics

Structural proteomics is the application of protein chemistry and modern mass spectrometric techniques to problems such as the characterization of protein structures and assemblies and the detailed determination of protein-protein interactions. The techniques used in structural proteomics include crosslinking, photoaffinity labeling, limited proteolysis, chemical protein modification and hydrogen/deuterium exchange, all followed by mass spectrometric analysis. None of these methods alone can provide complete structural information, but a "combination" of these complementary approaches can be used to provide enough information for answering important biological questions. Structural proteomics can help to determine, for example, the detailed structure of the interfaces between proteins that may be important drug targets and the interactions between proteins and ligands. In this review, we have tried to provide a brief overview of structural proteomics methodologies, illustrated with examples from our laboratory and from the literature.     

Proteomics study of rice embryogenesis: discovery of the embryogenesis-dependent globulins

The plant embryo is the germination center of the seed. How an embryo forms during seed maturation remains unclear, especially in the case of monocotyledonous plants. Generally, the complex processes of embryogenesis result from the action of a coordinated network of genes. Thus, a large-scale survey of changes in protein abundance during embryogenesis is an effective approach to study the molecular events of embryogenesis. In this study, two-dimensional gel electrophoresis (2DE) was applied to separate rice embryo proteins collected during the three phases of embryogenesis: 6 days after pollination (DAP), 12 DAP, and 18 DAP. We then employed matrix-assisted laser desorption-ionization time of flight/time of flight mass spectrometry(MALDI TOF/TOF MS) to identify the phase-dependent differential 2DE spots. A total of 66 spots were discovered to be regulated during embryogenesis, and of these spots, 53 spots were identified. These proteins were further categorized into several functional classes, including storage, embryo development, stress response, glycolysis, and protein metabolism. Intriguingly, the major differential spots originated from three globulins. We further examined the possible mechanism underlying the globulins' multiple forms using Western blotting, proteolysis, and blue native gel electrophoresis techniques and found that the multiple forms of globulins were produced as a result of enhanced proteolysis during embryogenesis, indicating that these globulin forms may serve as chaperone proteins participating in the formation of multiple protein complexes during embryogenesis.     

Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions

Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA The study of isolated protein complexes has greatly benefited from recent advances in mass spectrometry instrumentation and quantitative, isotope labeling techniques. The comprehensive characterization of protein complex components and quantification of their relative abundance relies heavily upon maximizing protein and peptide sequence information obtained from MS and tandem MS studies. Recent work has shown that using a metalloendopeptidase, Lys-N, for proteomic analysis of biological protein mixtures produces complementary protein sequence information compared with trypsin digestion alone. Here, we have investigated the suitability of Lys-N proteolysis for use with MALDI mass spectrometry to characterize the yeast Arp2 complex and E. coli PAP I protein interactions. Although Lys-N digestion resulted in an average decrease in protein sequence coverage of ∼30% compared with trypsin digestion, CID analysis of singly-charged Lys-N peptides yielded a more extensive b-ions series compared with complementary tryptic peptides. Taking advantage of this improved fragmentation pattern, we utilized differential ¹⁵N/¹⁴N guanidination of Lys-N peptides and MALDI-MS/MS analysis to relatively quantify the changes in PAP I associations due to deletion of sprE, previously shown to regulate PAP I-dependent polyadenylation. Overall, this Lys-N/guanidination integrative approach is applicable for functional proteomic studies utilizing MALDI mass spectrometry analysis, as it provides an effective and economical mean for relative quantification of proteins in conjunction with increased sensitivity of detection and fragmentation efficiency.     

Determination of protein adducts of organophosphorus nerve agents in blood plasma

A comparative study of the efficiency of procedures for the determination of the biomarkers of organophosphorus agents (OPAs) in blood plasma was performed. It was found that the gas chromatography–mass spectrometry determination of OPAs reactivated from the composition of protein adducts is a rapid method for the detection of exposure to OPAs. The liquid chromatography–mass spectrometry determination of phosphonylated butyrylcholinesterase and albumin fragments modified with OPA residues provides an opportunity to perform more sensitive and retrospective analysis. The tyrosine adducts of OPAs with serum albumin and other blood plasma proteins are not prone to dealkylation in the course of aging; in the series of the test markers, they possess the greatest diagnostic value because they make it possible to determine the precise structure a toxic agent after the longest time interval after exposure. The tentative limit of detection of OPA markers varies from 0.1 to 1.0 ng/mL.     

Top-down mass spectrometry: recent developments, applications and perspectives

Top-down mass spectrometry is an emerging approach for the analysis of intact proteins. The term was coined as a contrast with the better-established, bottom-up strategy for analysis of peptide fragments derived from digestion, either enzymatically or chemically, of intact proteins. Although the term top-down originates from proteomics, it can also be applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein assemblies or complexes. Traditionally, mass spectrometry has usually started with intact molecules, and in this regard, top-down approaches reflect the spirit of mass spectrometry. This article provides an overview of the methodologies in top-down mass spectrometry and then reviews applications covering protein posttranslational modifications, protein biophysics, DNAs/RNAs, and protein assemblies. Finally, challenges and future directions are discussed.     

Utility of N-peracetylation of proteins for their structure determination by mass spectrometry

Acetylation of the animo groups (N-terminus and lysine) of proteins before enzymatic or chemical cleavage was explored as an approach to provide additional information in the course of the determination of amino acid sequences. The major advantage is the ability to differentiate glutamine from lysine, because only the latter is acetylated and thus increases in mass by 42 Da. Horse heart cytochrome c could be fully N-actetylated and even on prolonged digestion with chymotrypsin underwent very little tryptic cleavage, in contrast to the native protein where this side reaction is extensive. Sperm whale myoglobin is more difficult to acetylate, but even at 40%–50% average acetylation, all 19 lysines could be identified unambiguously. A proteolytic digest of acetylated protein is thus a useful component of strategies for the determination of the primary structure of proteins by tandem mass spectrometry.     

Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements

The three-dimensional conformation of a protein is central to its biological function. The characterisation of aspects of three-dimensional protein structure by mass spectrometry is an area of much interest as the gas-phase conformation, in many instances, can be related to that of the solution phase. Travelling wave ion mobility mass spectrometry (TWIMS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIMS under denaturing and near-physiological solvent conditions and cross-sections estimated for the charge states observed. Estimates of collision cross-sections were obtained with reference to known standards with published cross-sections. Estimated cross-sections were compared with values from published X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy structures. The cross-section measured by ion mobility mass spectrometry varies with charge state, allowing the unfolding transition of proteins in the gas phase to be studied. Cross-sections estimated experimentally for proteins studied, for charge states most indicative of native structure, are in good agreement with measurements calculated from published X-ray and NMR structures. The relative stability of gas-phase structures has been investigated, for the proteins studied, based on their change in cross-section with increase in charge. These results illustrate that the TWIMS approach can provide data on three-dimensional protein structures of biological relevance.     

Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides provides powerful insight into the quaternary structure of protein complexes. Mixed-isotope cross-linking (a method for distinguishing intermolecular cross-links) was coupled with liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) to provide an additional separation dimension to the traditional cross-linking approach. This method produced multiplet m/z peaks that are aligned in the IMS drift time dimension and serve as signatures of intermolecular cross-linked peptides. We developed an informatics tool to use the amino acid sequence information inherent in the multiplet spacing for accurate identification of the cross-linked peptides. Because of the separation of cross-linked and non-cross-linked peptides in drift time, our LC-IMS-MS approach was able to confidently detect more intermolecular cross-linked peptides than LC-MS alone.      

A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry

Mass spectrometric analysis of wild-type proteins that have been covalently modified by bifunctional cross-linking reagents and then digested proteolytically can be used to obtain low-resolution distance constraints, which can be useful for protein structure determination. Limitations of this approach include time-consuming separation steps, such as the separation of internally cross-linked protein monomers from covalent dimers, and a susceptibility to artifacts due to low levels of natural and man-made peptide modifications that can be mistaken for cross-linked species. The results presented here show that when a crude cross-linked protein mixture is injected into an electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) instrument, the cross-link positions can be localized by fragmentation and mass spectrometry on the 'gas-phase purified' singly internally cross-linked monomer. Our results show that reaction of ubiquitin with the homobifunctional lysine-lysine cross-linking reagent dissuccinimidyl suberate (DSS) resulted in two cross-links consistent with the known ubiquitin tertiary structure (K6-K11 and K48-K63). Because no protein or peptide chemistry steps are needed, other than the initial cross-linking, this new top down approach appears well suited for high-throughput experiments with multiple cross-linkers and reaction conditions. Published in 2002 by John Wiley & Sons, Ltd.