Characterization of proteins by ambient mass spectrometry

Proteins play important roles in living systems and are topics of many fundamental and applied research projects. With the introduction of electrospray ionization and matrix‐assisted laser desorption/ionization for analysis of biomacromolecules in the late 1980s, mass spectrometry has become an important tool for characterization of proteins. Characterization of proteins in raw samples by these mass spectrometric techniques, however, usually requires extensive sample pretreatment. Ambient ionization techniques are new mass spectrometric techniques that allow direct analysis of samples with no or little sample preparation. Can these techniques facilitate or even eliminate sample preparation for mass spectrometric analysis of proteins? Apart from sample preparation, do these techniques offer any new features for characterization of proteins as compared with conventional ESI or MALDI? Recent advances in characterization of proteins by ambient mass spectrometry are summarized and commented in this article. © 2011 Wiley Periodicals, Inc. Mass Spec Rev 31:437–447, 2012     

Microorganism characterization by single particle mass spectrometry

In recent years a major effort by several groups has been undertaken to identify bacteria by mass spectrometry at the single cell level. The intent of this review is to highlight the recent progress made in the application of single particle mass spectrometry to the analysis of microorganisms. A large portion of the review highlights improvements in the ionization and mass analysis of bio-aerosols, or particles that contain biologically relevant molecules such as peptides or proteins. While these are not direct applications to bacteria, the results have been central to a progression toward single cell mass spectrometry. Developments in single particle matrix-assisted laser desorption/ionization (MALDI) are summarized. Recent applications of aerosol laser desorption/ionization (LDI) to the analysis of single microorganisms are highlighted. Successful applications of off-line and on-the-fly aerosol MALDI to microorganism detection are discussed. Limitations to current approaches and necessary future achievements are also addressed.     

Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions

Closely related to studying the function of a protein is the analysis of its three-dimensional structure and the identification of interaction sites with its binding partners. An alternative approach to the high-resolution methods for three-dimensional protein structure analysis, such as X-ray crystallography and NMR spectroscopy, consists of covalently connecting two functional groups of the protein(s) under investigation. The location of the created cross-links imposes a distance constraint on the location of the respective side chains and allows one to draw conclusions on the three-dimensional structure of the protein or a protein complex. Recently, chemical cross-linking of proteins has been combined with a mass spectrometric analysis of the created cross-linked products. This review article describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the different available strategies that employ chemical cross-linking and different mass spectrometric techniques. The challenges for mass spectrometry caused by the enormous complexity of the cross-linking reaction mixtures are emphasized. The various approaches described in the literature to facilitate the mass spectrometric detection of cross-linked products as well as computer software for data analyses are reviewed.     

Assessment of protein expression by means of 2-D gel electrophoresis with and without mass spectrometry

Careful examination of current literature, particularly over the last 5 years, reveals a wide range of approaches for the relative quantification of protein expression in cells, tissues, and body fluids. In view of such an observation, it is reasonable to ask whether researchers need new methods, or whether it is more productive to optimize and tune already existing ones. It is generally agreed that none of the existing methodologies on its own can give a full account of protein expression in a complex medium; this limitation, however, has not prevented the use of existing methods to provide valuable information on a wide range of proteins, where their expression has been correlated to certain pathologies and/or to pharmacological, genetic, or environmental factors. In the present work, an attempt is made to review the application of one of these methodologies, namely two-dimensional polyacrylamide gel electrophoresis on its own or in conjunction with mass spectrometry, to assess protein expression, particularly when such expression can be correlated to certain pathologies.     

Automated protein identification by tandem mass spectrometry: issues and strategies

Protein identification by tandem mass spectrometry (MS/MS) is key to most proteomics projects and has been widely explored in bioinformatics research. Obtaining good and trustful identification results has important implications for biological and clinical work. Although well matured, automated software identification of proteins from MS/MS data still faces a number of obstacles due to the complexity of the proteome or procedural issues of mass spectrometry data acquisition. Expected or unexpected modifications of the peptide sequences, polymorphisms, errors in databases, missed or non-specific cleavages, unusual fragmentation patterns, and single MS/MS spectra of multiple peptides of the same m/z are so many pitfalls for identification algorithms. A lot of research work has been carried out in recent years that yielded new strategies to handle a number of these issues. Multiple MS/MS identification algorithms are now available or have been theoretically described. The difficulty resides in choosing the most adapted method for each type of spectra being identified. This review presents an overview of the state-of-the-art bioinformatics approaches to the identification of proteins by MS/MS to help the reader doing the spade work of finding the right tools among the many possibilities offered.     

Investigation of intact protein complexes by mass spectrometry

Mass spectrometry has grown in recent years to a well-accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non-covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non-covalent protein-protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights.     

Identification and characterization of molecular targets of natural products by mass spectrometry

Natural products, and their derivatives and mimics, have contributed to the development of important therapeutics to combat diseases such as infections and cancers over the past decades. The value of natural products to modern drug discovery is still considerable. However, its development is hampered by a lack of a mechanistic understanding of their molecular action, as opposed to the emerging molecule‐targeted therapeutics that are tailored to a specific protein target(s). Recent advances in the mass spectrometry‐based proteomic approaches have the potential to offer unprecedented insights into the molecular action of natural products. Chemical proteomics is established as an invaluable tool for the identification of protein targets of natural products. Small‐molecule affinity selection combined with mass spectrometry is a successful strategy to “fish” cellular targets from the entire proteome. Mass spectrometry‐based profiling of protein expression is also routinely employed to elucidate molecular pathways involved in the therapeutic and possible toxicological responses upon treatment with natural products. In addition, mass spectrometry is increasingly utilized to probe structural aspects of natural products–protein interactions. Limited proteolysis, photoaffinity labeling, and hydrogen/deuterium exchange in conjunction with mass spectrometry are sensitive and high‐throughput strategies that provide low‐resolution structural information of non‐covalent natural product–protein complexes. In this review, we provide an overview on the applications of mass spectrometry‐based techniques in the identification and characterization of natural product–protein interactions, and we describe how these applications might revolutionize natural product‐based drug discovery. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:126–155, 2010     

Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research

Progress in proteomics research has led to a demand for powerful analytical tools with high separation efficiency and sensitivity for confident identification and quantification of proteins, posttranslational modifications, and protein complexes expressed in cells and tissues. This demand has significantly increased interest in capillary electrophoresis‐mass spectrometry (CE‐MS) in the past few years. This review provides highlights of recent advances in CE‐MS for proteomics research, including a short introduction to top‐down mass spectrometry and native mass spectrometry (native MS), as well as a detailed overview of CE methods. Both the potential and limitations of these methods for the analysis of proteins and peptides in synthetic and biological samples and the challenges of CE methods are discussed, along with perspectives about the future direction of CE‐MS. @ 2019 Wiley Periodicals, Inc. Mass Spec Rev 00:1–16, 2019.     

Protein sequence information by matrix-assisted laser desorption/ionization in-source decay mass spectrometry

Proteins from biological samples are often identified by mass spectrometry (MS) with the two following "bottom-up" approaches: peptide mass fingerprinting or peptide sequence tag. Nevertheless, these strategies are time-consuming (digestion, liquid chromatography step, desalting step), the N- (or C-) terminal information often lacks and post-translational modifications (PTMs) are hardly observed. The in-source decay (ISD) occurring in a matrix assisted laser desorption/ionization (MALDI) source appears an interesting analytical tool to obtain N-terminal sequence, to identify proteins and to characterize PTMs by a "top-down" strategy. The goal of this review deals with the usefulness of the ISD technique in MALDI source in proteomics fields. In the first part, the ISD principle is explained and in the second part, the use of ISD in proteomic studies is discussed for protein identification and sequence characterization.     

Mass spectrometry in the biosynthetic and structural investigation of lignins

Lignin, a resistant cell-wall constituent of all vascular plants that consists of ether and carbon-linked methoxyphenols, is still far from being structurally described in detail. The main problem in its structural elucidation is the difficulty of isolating lignin from other wood components without damaging lignin itself. Furthermore, the high number and variegated forms of linkages that occur between the monomeric units and the chemical resistance of certain ether bonds limit the extent to which analytical and degradation procedures can be used to elucidate the lignin structure. Most of our present knowledge about the molecular structure of lignin is based on the analysis of monomers, dimers or, at the most, tetramers of degraded isolated lignins. Mass spectrometry (MS), which offers advantages in terms of speed, specificity, and sensitivity, has revealed to be a very powerful technique in the structural elucidation of lignins, in combination with the great number of chemical and thermal degradation methods available in the study of lignin. Moreover, the recent development of new ionization techniques in MS-electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS-has provided new possibilities to also analyze the undegraded lignin macromolecule.     

Mass spectrometry of peptides and proteins from human blood

It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post‐translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post‐translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ～1 ng/mL. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:685–732, 2011     

Peptide and protein drugs: the study of their metabolism and catabolism by mass spectrometry

Peptide and protein drugs have evolved in recent years into mainstream therapeutics, representing a significant portion of the pharmaceutical market. Peptides and proteins exhibit highly diverse structures, broad biological activities as hormones, neurotransmitters, structural proteins, metabolic modulators and therefore have a significant role as both therapeutics and biomarkers. Understanding the metabolism of synthetic or biotechnologically derived peptide and protein drugs is critical for pharmaceutical development as metabolism has a significant impact on drug efficacy and safety. Although the same principles of pharmacokinetics and metabolism of small molecule drugs apply to peptide and protein drugs, there are few notable differences. Moreover, the study of peptide and protein drug metabolism is a rather complicated process which requires sophisticated analytical techniques, and mass spectrometry based approaches have provided the capabilities for efficient and reliable quantification, characterization, and metabolite identification. This review article will focus on the current use of mass spectrometry for the study of the metabolism of peptide and protein drugs.     

Structural characterization of bacterial lipopolysaccharides with mass spectrometry and on‐ and off‐line separation techniques

The focus of this review is the application of mass spectrometry to the structural characterization of bacterial lipopolysaccharides (LPSs), also referred to as “endotoxins,” because they elicit the strong immune response in infected organisms. Recently, a wide variety of MS‐based applications have been implemented to the structure elucidation of LPS. Methodological improvements, as well as on‐ and off‐line separation procedures, proved the versatility of mass spectrometry to study complex LPS mixtures. Special attention is given in the review to the tandem mass spectrometric methods and protocols for the analyses of lipid A, the endotoxic principle of LPS. We compare and evaluate the different ionization techniques (MALDI, ESI) in view of their use in intact R‐ and S‐type LPS and lipid A studies. Methods for sample preparation of LPS prior to mass spectrometric analysis are also described. The direct identification of intrinsic heterogeneities of most intact LPS and lipid A preparations is a particular challenge, for which separation techniques (e.g., TLC, slab‐PAGE, CE, GC, HPLC) combined with mass spectrometry are often necessary. A brief summary of these combined methodologies to profile LPS molecular species is provided. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:90–117, 2013     

Liquid chromatography combined with mass spectrometry for 13C isotopic analysis in life science research

Among the different disciplines covered by mass spectrometry, measurement of (13)C/(12)C isotopic ratio crosses a large section of disciplines from a tool revealing the origin of compounds to more recent approaches such as metabolomics and proteomics. Isotope ratio mass spectrometry (IRMS) and molecular mass spectrometry (MS) are the two most mature techniques for (13)C isotopic analysis of compounds, respectively, for high and low-isotopic precision. For the sample introduction, the coupling of gas chromatography (GC) to either IRMS or MS is state of the art technique for targeted isotopic analysis of volatile analytes. However, liquid chromatography (LC) also needs to be considered as a tool for the sample introduction into IRMS or MS for (13)C isotopic analyses of non-volatile analytes at natural abundance as well as for (13)C-labeled compounds. This review presents the past and the current processes used to perform (13)C isotopic analysis in combination with LC. It gives particular attention to the combination of LC with IRMS which started in the 1990's with the moving wire transport, then subsequently moved to the chemical reaction interface (CRI) and was made commercially available in 2004 with the wet chemical oxidation interface (LC-IRMS). The LC-IRMS method development is also discussed in this review, including the possible approaches for increasing selectivity and efficiency, for example, using a 100% aqueous mobile phase for the LC separation. In addition, applications for measuring (13)C isotopic enrichments using atmospheric pressure LC-MS instruments with a quadrupole, a time-of-flight, and an ion trap analyzer are also discussed as well as a LC-ICPMS using a prototype instrument with two quadrupoles.     

Studying the interactome with the yeast two-hybrid system and mass spectrometry

Protein interactions are crucial to the life of a cell. The analysis of such interactions is allowing biologists to determine the function of uncharacterized proteins and the genes that encode them. The yeast two-hybrid system has become one of the most popular and powerful tools to study protein-protein interactions. With the advent of proteomics, the two-hybrid system has found a niche in interactome mapping. However, it is clear that only by combining two-hybrid data with that from complementary approaches such as mass spectrometry (MS) can the interactome be analyzed in full. This review introduces the yeast two-hybrid system to those unfamiliar with the technique, and discusses how it can be used in combination with MS to unravel the network of protein interactions that occur in a cell.     

Environmental and food applications of LC-tandem mass spectrometry in pesticide-residue analysis: an overview

An overview is given on pesticide-residue determination in environmental and food samples by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Pesticides comprise a large number of substances that belong to many completely different chemical groups, the only common characteristic is that they are effective against pests. They still constitute a challenge in MS because there is no collective pathway for fragmentation. A brief introduction to the theory of tandem MS permits a discussion of which parameters influence the ionization efficiency when the ions are subjected to different actions. Emphasis is placed on the different tandem MS instruments: triple and ion-trap quadrupoles, and hybrid quadrupole time-of-flight (Q-TOF), including advantages and drawbacks, typical detection limits, and ion signals at low concentrations. The instrumental setup, as well as LC and mass spectrometric experimental conditions, must be carefully selected to increase the performance of the analytical system. The capacity of each instrument to provide useful data for the identification of pesticides, and the possibility to obtain structural information for the identification of target and non-target compounds, are discussed. Finally, sample preparation techniques and examples of applications are debated to reveal the potential of the current state-of-the-art technology, and to further promote the usefulness of tandem MS.     

Differentiation of isomeric amino acid residues in proteins and peptides using mass spectrometry

Characterization and differentiation of isomers in biological macromolecules using mass spectrometry is one of the most significant challenges facing scientists in the field. The capability of high‐resolution MS instruments along with the development of new fragmentation methods now provides the ability to indirectly differentiate between some isomers. This ability has enabled mass spectrometry to evolve into a multidisciplinary technique incorporating areas such as pharmaceutical research, proteomics, polymer science, medicine, environmental chemistry, and recently archeology. This article aims to review recent developments in mass spectrometry methodologies in the identification of structural and spatial isomers in biological macromolecules, such as aspartic acid and isoaspartic acid (Asp/IsoAsp), leucine and isoleucine (Leu/Ile), glutamic acid and γ‐glutamic acid, and D/L enantiomers. © 2012 Wiley Periodicals, Inc. Mass Spec Rev 31:609–625, 2012