Recent Developments In Mass Spectrometry Research Articles

Recent development of mass spectrometry and proteomics applications in identification and typing of bacteria.

Identification and typing of bacteria occupy a large fraction of time and work in clinical microbiology laboratories. With the certification of some MS platforms in recent years, more applications and tests of MS‐based diagnosis methods for bacteria identification and typing have been created, not only on well‐accepted MALDI‐TOF‐MS‐based fingerprint matches, but also on solving the insufficiencies of MALDI‐TOF‐MS‐based platforms and advancing the technology to areas such as targeted MS identification and typing of bacteria, bacterial toxin identification, antibiotics susceptibility/resistance tests, and MS‐based diagnostic method development on unique bacteria such as Clostridium and Mycobacteria. This review summarizes the recent development in MS platforms and applications in bacteria identification and typing of common pathogenic bacteria.

A new window into the molecular physiology of membrane proteins.

Integral membrane proteins comprise ∼25% of the human proteome. Yet, our understanding of their molecular physiology is still in its infancy. This can be attributed to two factors: the experimental challenges that arise from the difficult chemical nature of membrane proteins, and the unclear relationship between their activity and their native environment. New approaches are therefore required to address these challenges. Recent developments in mass spectrometry have shown that it is possible to study membrane proteins in a solvent-free environment and provide detailed insights into complex interactions, ligand binding and folding processes. Interestingly, not only detergent micelles but also lipid bilayer nanodiscs or bicelles can serve as a means for the gentle desolvation of membrane proteins in the gas phase. In this manner, as well as by direct addition of lipids, it is possible to study the effects of different membrane components on the structure and function of the protein components allowing us to add functional data to the least accessible part of the proteome.

Flexible membrane proteins: functional dynamics captured by mass spectrometry.

Membrane proteins are flexible molecular machines, responsible for the exchange of molecules in and out of the cell, which have evolved to perform specific and complex tasks with great efficiency. Obtaining accurate descriptions of their dynamics in the context of their function represents a major challenge for structural biology. Here we chart recent developments in mass spectrometry designed to characterize changes in the dynamics of membrane proteins in response to ligand binding or post-translational modifications. We focus on cooperative movements and structural changes across a range of timescales, from milliseconds to minutes, and highlight the contributions of mass spectrometry to our understanding of molecular mechanisms of diverse transmembrane processes.

Twenty years of protein interaction studies for biological function deciphering

Intensive methodological developments and technology innovation have been devoted to protein-protein interaction studies over 20years. Genetic indirect assays and sophisticated large scale biochemical analyses have jointly contributed to the elucidation of protein-protein interactions, still with a lot of drawbacks despite heavy investment in human resources and technologies. With the most recent developments in mass spectrometry and computational tools for studying protein content of complex samples, the initial goal of deciphering molecular bases of biological functions is now within reach. Here, we described the various steps of this process and gave examples of key milestones in this scientific story line. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

Protein profiling using mass spectrometry

This review summarizes briefly the methods and progresses of qualitative and quantitative mass-spectrometry based analysis on proteins expressed in living organisms. Systematic analysis of all gene-coding proteins of a biosample offers highly complementary information to genomics and yields new biological insight into diseases. The systematic technique by means of mass-spectrometry analysis has played a critical role in protein profiling, and can identify and quantify proteins with high throughput, high sensitivity and high accuracy. The review provides the recent developments of mass-spectrometry based proteomics in the past decades especially in China, and prospects the trend of further study as well.

Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD)

The identification of peptides presented by human leukocyte antigen (HLA) class I is tremendously important for the understanding of antigen presentation mechanisms under healthy or diseased conditions. Currently, mass spectrometry-based methods represent the best methodology for the identification of HLA class I-associated peptides. However, the HLA class I peptide repertoire remains largely unexplored because the variable nature of endogenous peptides represents difficulties in conventional peptide fragmentation technology. Here, we substantially enhanced (about threefold) the identification success rate of peptides presented by HLA class I using combined electron-transfer/higher-energy collision dissociation (EThcD), reporting over 12,000 high-confident (false discovery rate <1%) peptides from a single human B-cell line. The direct importance of such an unprecedented large dataset is highlighted by the discovery of unique features in antigen presentation. The observation that a substantial part of proteins is sampled across different HLA alleles, and the common occurrence of HLA class I nested sets, suggest that the constraints of HLA class I to comprehensively present the health states of cells are not as tight as previously thought. Our dataset contains a substantial set of peptides bearing a variety of posttranslational modifications presented with marked allele-specific differences. We propose that EThcD should become the method of choice in analyzing HLA class I-presented peptides.

Utility of computational structural biology in mass spectrometry.

Recent developments of mass spectrometry (MS) allow us to identify, estimate, and characterize proteins and protein complexes. At the same time, structural biology helps to determine the protein structure and its structure-function relationship. Together, they aid to understand the protein structure, property, function, protein-complex assembly, protein-protein interaction and dynamics. The present chapter is organized with illustrative results to demonstrate how experimental mass spectrometry can be combined with computational structural biology for detailed studies of protein's structures. We have used tumor differentiation factor protein/peptide as ligand and Hsp70/Hsp90 as receptor protein as examples to study ligand-protein interaction. To investigate possible protein conformation we will describe two proteins, lysozyme and myoglobin.

Red Blood Cell Lipidomics analysis through HPLC-ESI-qTOF: application to red blood cell storage

Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrices. In the present study we performed an on-line HPLC-microTOF-Q MS approach to the red blood cell (RBC) lipidome. We thus exploited bioinformatic tools for the interrogation of novel databases, such as LIPID MAPS. By means of ad hoc software suites for mass spectrometry-based metabolomics analyses, we could address the key biological issue of the RBC lipidome, within the framework of RBC storage for transfusion purposes. Samples were collected from subjects living in the province of Viterbo, where olive oil consumption represents a central aspect of the diet. On this ground, we could postulate a diet specific effect on the accumulation of lipid-specific storage lesions. The analyses yielded the tentative identification of a huge number of lipid molecules on the basis of accurate intact mass values and retention times, and MS/MS validation. This analytical workflow was exploited to consolidate existing knowledge on the RBC lipid composition and individuate statistically significant fluctuations of lipids throughout storage duration of RBC concentrates under blood bank conditions. Our analysis indicated ceramides, glycerophospholipids and sterols as key targets of RBC storage lesions to the lipidome, that will deserve further targeted investigations in the future. It also emerged how compositional analyses of the RBC lipidome might end up yielding different results on the basis of the background of the blood donor (i.e. diet), which might translate into region-specific lipidomic alterations over storage progression of RBC concentrates.

Determination of accurate protein monoisotopic mass with the most abundant mass measurable using high-resolution mass spectrometry

While recent developments in mass spectrometry enable direct evaluation of monoisotopic masses (Mmi) of smaller compounds, protein Mmi is mostly determined based on its relationship to average mass (Mav). Here, we propose an alternative approach to determining protein Mmi based on its correlation with the most abundant mass (Mma) measurable using high-resolution mass spectrometry. To test this supposition, we first empirically calculated Mmi and Mma of 6158 Escherichia coli proteins, which helped serendipitously uncover a linear correlation between these two protein masses. With the relationship characterized, liquid chromatography–mass spectrometry was employed to measure Mma of protein samples in its ion cluster with the highest signal in the mass spectrum. Generally, our method produces a short series of likely Mmi in 1-Da steps, and the probability of each likely Mmi is assigned statistically. It is remarkable that the mass error of this Mmi is as miniscule as a few parts per million, indicating that our method is capable of determining protein Mmi with high accuracy. Benefitting from the outstanding performance of modern mass spectrometry, our approach is a significant improvement over others and should be of great utility in the rapid assessment of protein primary structures.

Mass spectrometry: towards in vivo analysis of biological systems

In vivo analysis is of paramount importance in monitoring physiological processes that take place in living organisms. Mass spectrometry, an analytical technique with high speed, sensitivity and specificity, is indispensable in biochemical studies nowadays. However, traditional mass spectrometric techniques are of limited applicability in direct analysis of living organisms due to various constraints, e.g., the necessity of ionization of analytes under vacuum and perturbation of physiological functions of living organisms during analysis. Recent development of mass spectrometry, particularly the development of ambient ionization techniques, has opened the door for direct analysis of living organisms. These new mass spectrometric techniques have the features that the ionization processes take place under atmospheric pressure and no or only little sample preparation is required, thus are well suited for analysis of living specimens without significantly perturbing their physiological states. The role of these mass spectrometric techniques in in vivo analysis has been increasingly important in recent years and is expected to be further expanded in the future. In this review, the use of various mass spectrometric techniques in in vivo analysis of biological systems is summarized and the prospects are discussed.

Linking structural change with functional regulation—insights from mass spectrometry

A wide range of biophysical approaches has been applied to structural biology, all with the same overall goal-to understand the molecular machines that allow cells to function. While knowledge of the identity and composition of component protein subunits is an important foundation for understanding these macromolecular complexes it has become increasingly clear that knowledge of the exact composition alone is insufficient for understanding dynamic interactions and regulatory mechanisms. In this review we focus on recent developments of mass spectrometry (MS) that allow us to unravel the functional 'secrets' of non-covalent molecular machines.

MD For MS: Simulations of the Transfer of Membrane Protein-Detergent Complexes from Solution to Vacuum in Electrospray Ionisation Mass Spectrometry (ESI-MS)

Recent developments in mass spectrometry have allowed membrane proteins in complex with detergent micelles to be studied in the gas phase - previously thought only possible for soluble proteins. This development paves the way for future studies of membrane protein stability, structure and oligomerisation. The gas phase environment is very different to that of a cell membrane, and questions have been raised over the conformation of proteins in the gas phase. Molecular dynamics (MD) simulations have been used extensively to explore the evolution of gas phase structures of soluble proteins, with some evidence that the native state may be stabilised in vacuum (although the timescale of this stability remains unclear). However, membrane protein-detergent complexes have been less well studied by simulations. We have used a combination of non-equilibrium and equilibrium MD simulations to elucidate some of the processes that occur during transition of a protein-detergent complex from aqueous solution to vacuum (thus mimicking electrospray ionisation). We compare two membrane protein architectures (an α-helical bundle vs a β-barrel) and different detergents with respect to protein-detergent complex dynamics and stability.

Analytical aspects in doping control: Challenges and perspectives

Since the first anti-doping tests in the 1960s, the analytical aspects of the testing remain challenging. The evolution of the analytical process in doping control is discussed in this paper with a particular emphasis on separation techniques, such as gas chromatography and liquid chromatography. These approaches are improving in parallel with the requirements of increasing sensitivity and selectivity for detecting prohibited substances in biological samples from athletes. Moreover, fast analyses are mandatory to deal with the growing number of doping control samples and the short response time required during particular sport events. Recent developments in mass spectrometry and the expansion of accurate mass determination has improved anti-doping strategies with the possibility of using elemental composition and isotope patterns for structural identification. These techniques must be able to distinguish equivocally between negative and suspicious samples with no false-negative or false-positive results. Therefore, high degree of reliability must be reached for the identification of major metabolites corresponding to suspected analytes. Along with current trends in pharmaceutical industry the analysis of proteins and peptides remains an important issue in doping control. Sophisticated analytical tools are still mandatory to improve their distinction from endogenous analogs. Finally, indirect approaches will be discussed in the context of anti-doping, in which recent advances are aimed to examine the biological response of a doping agent in a holistic way.

A Quantitative Perspective on Hydrophobic Interactions in the Gas-Phase

Mass spectrometry has become a powerful tool for determining the composition, stoichiometry, subunit interactions, and architectural organization of non-covalent protein complexes. The vast majority of assemblies studied so far by this approach are those that contain a sufficient amount of electrostatic interactions and hydrogen bonds that can survive the transition from solution to the gas-phase and maintain the structural features of the vaporized ions. An intriguing question that naturally arises is whether mass spectrometry can also be harnessed for the study of molecular systems dominated by non-polar interactions. Here we address this issue and discuss the fate of hydrophobic complexes in the absence of bulk water molecules. We emphasize the progress that has been accomplished in this field that is moving towards the analysis of larger and more complex hydrophobic systems. We attribute this advance to recent developments of mass spectrometry and its application to non-covalent complexes in general, and to the understanding of experimental and biochemical conditions for the preservation of hydrophobic interactions in particular. Furthermore, we discuss the ability of mass spectrometry to serve as a quantitative tool for assessing the strength, binding energies, and stoichiometries of hydrophobic interactions. Overall, we aim to stimulate research in this area and to establish mass spectrometry as a tool for analyzing hydrophobic interactions within complex biological systems.

Improving the Biomarker Pipeline

Proteomics has evolved over the past 10 years from a method providing mainly qualitative information—protein identification and modification site determination—to a more quantitative technique. Until quite recently, quantitative proteomics was focused on relative quantification for biomarker discovery. Biomarker discovery, by definition, must take a “shotgun” approach, which usually involves a 2-dimensional separation (1). Unfortunately, this approach also means that the techniques used for biomarker discovery are both time-consuming and costly; consequently, few potential biomarkers have progressed beyond the discovery phase. Liquid chromatography–tandem mass spectrometry quantitative proteomics techniques designed for biomarker discovery, such as isobaric tags for relative and absolute quantification (iTRAQ)2 2), can take 1–2 weeks for a comparison of 8 samples. One bottleneck is the low throughput of “conventional” proteomics techniques, which usually use low-flow-rate nanoscale liquid chromatography and long analysis times for the highest possible detection of biomarkers and exhaustive coverage of the proteome. Today, there is a great deal of interest in the next phase in the biomarker pipeline, biomarker validation and biomarker verification, which must be done before clinical evaluation can be performed (3). The success of mass spectrometry–based comparative proteomics studies with relative quantitative methods has led to the discovery of thousands of potential biomarkers that now need to be verified and validated. Instead of the in-depth analysis of a few samples, however, the verification/validation phases involve the absolute quantification and targeted analysis of a large number of samples. Different methods can be used for quantification because the protein targets are already known. Today, ELISA assays are used for biomarker verification and validation of targeted proteins. Times are changing, however. Recent developments in mass spectrometry–based proteomics have led to the development of higher-throughput targeted analyses that still retain the superior specificity obtained with gas-phase sequence determination. These analyses focus not …

Methodological advances in sperm proteomics

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims to identify the proteins that compose the sperm cell and the study of their function. Recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and study sperm proteins. Catalogues of hundreds to thousands of spermatozoan proteins in human and in model species are becoming available setting up the basis for subsequent research, diagnostic applications and the development of specific treatments. A wide range of MS techniques are also rapidly becoming available for researchers. The present review summarises the different methodological options to study the sperm cell using MS and to provide a summary of some of the ongoing proteomic studies.

Proteomics and the genetics of sperm chromatin condensation

Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies.

Nano-HPLC–MS analysis of phospholipids in cerebrospinal fluid of Alzheimer’s disease patients—a pilot study

There is emerging evidence that lipids play an important role in many neurodegenerative processes, for example in Alzheimer's disease (AD). Although different lipid alterations in the AD brain have been reported, there have only been very few investigations of lipid changes in the cerebrospinal fluid (CSF). Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrixes. In this study we developed an on-line HPLC-MS method for phospholipid profiling in the CSF based on nano-HPLC separation using an Amide column and detection with electrospray (ESI) quadrupole-time of flight (QTOF) MS. We achieved good separation, reproducibility, and sensitivity in monitoring of the major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), and sphingomyelin (SM) in CSF. To emphasize the applicability of the method, a pilot study was performed on a group of CSF samples (N = 16) from individuals with probable AD and non-demented controls. We observed a statistically significant increase of SM levels (24.3 ± 2.4%) in CSF from probable AD individuals vs. controls. Our findings indicate that SM levels in the CSF could potentially provide a new lead in AD biomarker research, and show the potential of the method for disease-associated CSF phospholipid screening.

Mass Spectrometry—based Metabolomics, Analysis of Metabolite-Protein Interactions, and imaging

Our understanding of biology has been greatly improved through recent developments in mass spectrometry, which is providing detailed information on protein and metabolite composition as well as protein-metabolite interactions. The high sensitivity and resolution of mass spectrometry achieved with liquid or gas chromatography allows for detection and quantification of hundreds to thousands of molecules in a single measurement. Where homogenization-based sample preparation and extraction methods result in a loss of spatial information, mass spectrometry imaging technologies provide the in situ distribution profiles of metabolites and proteins within tissues. Mass spectrometry-based analysis of metabolite abundance, protein-metabolite interactions, and spatial distribution of compounds facilitates the high-throughput screening of biochemical reactions, the reconstruction of metabolic networks, biomarker discovery, determination of tissue compositions, and functional annotation of both proteins and metabolites.

Structure of glycosylated Cu/Zn-superoxide dismutase from Kluyveromyces yeast NBIMCC 1984

The primary structure of Cu/Zn-superoxide dismutase from Kluyveromyces marxianus NBIMCC 1984 was elucidated by N-terminal sequence analysis of the intact protein and by determination of the amino acid sequences of tryptic peptides by MALDI–TOF–TOF tandem mass spectrometry. The molecular mass of one subunit of the homodimer SOD, containing 152 amino acid residues, was calculated to be 15858.3 Da while a value of 17096.63 Da was obtained by MALDI–TOF MS. This difference is explained by the presence of N-glycosylation of one linkage site, -Asn-Ile/Leu-Thr-, and a glycan chain with the structure Hex 5 GlcNAc 2. Glycosylation of K. marxianus superoxide dismutase is a post-translational modification. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins. Therefore, in this paper, we introduce a covalent modification of Cu/Zn-SOD from K. marxianus NBIMCC 1984, by analysis of the enzymatic liberated N-glycan from the enzyme using MALDI–TOF and tandem mass spectrometry on a Q-Trap mass spectrometer. This is the first report of the structure of the oligosaccharide of a naturally-glycosylated superoxide dismutase, determined by mass spectrometry.