Functional genomics by mass spectrometry

Systematic analysis of the function of genes can take place at the oligonucleotide or protein level. The latter has the advantage of being closest to function, since it is proteins that perform most of the reactions necessary for the cell. For most protein based ('proteomic') approaches to gene function, mass spectrometry is the method of choice. Mass spectrometry can now identify proteins with very high sensitivity and medium to high throughput. New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification. New approaches include the isotopic labeling of proteins to obtain accurate quantitative data by mass spectrometry, methods to analyze peptides derived from crude protein mixtures and approaches to analyze large numbers of intact proteins by mass spectrometry directly. Examples from this laboratory illustrate biological problem solving by modern mass spectrometric techniques. These include the analysis of the structure and function of the nucleolus and the analysis of signaling complexes.     

Flow field-flow fractionation: a pre-analytical method for proteomics

Proteome analysis requires a comprehensive approach including high-performance separation methods, mass spectrometric analysis, and bioinformatics. While recent advances in mass spectrometry (MS) have led to remarkable improvements in the ability to characterize complex mixtures of biomolecules in proteomics, a proper pre-MS separation step of proteins/peptides is still required. The need of high-performance separation and/or isolation/purification techniques of proteins is increasing, due to the importance of proteins expressed at extremely low levels in proteome samples. In this review, flow field-flow fractionation (F4) is introduced as a complementary pre-analytical separation method for protein separation/isolation, which can be effectively utilized for proteomic research. F4 is a set of elution-based techniques that are capable of separating macromolecules by differences in diffusion coefficient and, therefore, in hydrodynamic size. F4 provides protein separation without surface interaction of the analyte with packing or gel media. Separation is carried out in an open channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly-applied, secondary flow of the fluid. Therefore, biological analytes such as proteins can be kept under a bio-friendly environment without losing their original structural configuration. Moreover, proteins fractionated on a size/shape basis can be readily collected for further characterization or proteomic analysis by MS using, for instance, either on-line or off-line methods based on electrospray ionization (ESI) or matrix-assisted laser desorption-ionization (MALDI). This review focuses on the advantages of F4 compared to most-assessed separation/isolation techniques for proteomics, and on selected applications based on size-dependent proteome separation. New method developments based on the hyphenation of F4 with on-line or off-line MS, and with other separation methods such as capillary isoelectric focusing (CIEF) are also described.     

Review of application of mass spectrometry for analyses of anterior eye proteome

Proteins have important functional roles in the body, which can be altered in disease states. The eye is a complex organ rich in proteins; in particular, the anterior eye is very sophisticated in function and is most commonly involved in ophthalmic diseases. Proteomics, the large scale study of proteins, has greatly impacted our knowledge and understanding of gene function in the post-genomic period. The most significant breakthrough in proteomics has been mass spectrometric identification of proteins, which extends analysis far beyond the mere display of proteins that classical techniques provide. Mass spectrometry functions as a "mass analyzer" which simplifies the identification and quantification of proteins extracted from biological tissue. Mass spectrometric analysis of the anterior eye proteome provides a differential display for protein comparison of normal and diseased tissue. In this article wepresent the key proteomic findings in the recent literature related to the cornea, aqueous humor, trabecular meshwork, iris, ciliary body and lens. Through this we identified unique proteins specific to diseases related to the anterior eye.     

Deepening our understanding of HDL proteome

ABSTRACT

Introduction: High-density lipoprotein (HDL) particles are heterogeneous and their proteome is complex and distinct from HDL cholesterol. However, it is largely unknown whether HDL proteins are associated with cardiovascular protection.

Areas covered: HDL isolation techniques and proteomic analyses are reviewed. A list of HDL proteins reported in 37 different studies was compiled and the effects of different isolation techniques on proteins attributed to HDL are discussed. Mass spectrometric techniques used for HDL analysis and the need for precise and robust methods for quantification of HDL proteins are discussed.

Expert opinion: Proteins associated with HDL have the potential to be used as biomarkers and/or help to understand HDL functionality. To achieve this, large cohorts must be studied using precise quantification methods. Key factors in HDL proteome quantification are the isolation methodology and the mass spectrometry technique employed. Isolation methodology affects what proteins are identified in HDL and the specificity of association with HDL particles needs to be addressed. Shotgun proteomics yields imprecise quantification, but the majority of HDL studies relied on this approach. Few recent studies used targeted tandem mass spectrometry to quantify HDL proteins, and it is imperative that future studies focus on the application of these precise techniques.     

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.     

Mass Spectrometry-Based Approaches Toward Absolute Quantitative Proteomics

Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques for stable isotope labeling developed over the past decade, it is now routinely used as a method for relative quantification to provide valuable information on alteration of protein abundance in a proteome-wide scale. More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides. On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides. Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.Key Words: Quantitative proteomics, mass spectrometry, absolute quantification, stable isotope labeling, label-free.     

Mass spectrometry-based proteomics for translational research: a technical overview

Mass spectrometry-based investigation of clinical samples enables the high-throughput identification of protein biomarkers. We provide an overview of mass spectrometry-based proteomic techniques that are applicable to the investigation of clinical samples. We address sample collection, protein extraction and fractionation, mass spectrometry modalities, and quantitative proteomics. Finally, we examine the limitations and further potential of such technologies. Liquid chromatography fractionation coupled with tandem mass spectrometry is well suited to handle mixtures of hundreds or thousands of proteins. Mass spectrometry-based proteome elucidation can reveal potential biomarkers and aid in the development of hypotheses for downstream investigation of the molecular mechanisms of disease.     

Advances in mass spectrometry for proteome analysis

The most demanding problems in proteomics continue to challenge modern mass spectrometry. Recent developments in instrument design have led to lower limits of detection, while new ion activation techniques and improved understanding of gas-phase ion chemistry have enhanced the capabilities of tandem mass spectrometry for peptide and protein structure elucidation. Future developments must address the., understanding of protein-protein interactions and the characterisation of the dynamic proteome.     

Classification and identification of bacteria using mass spectrometry-based proteomics

Timely classification and identification of bacteria is of vital importance in many areas of public health. Mass spectrometry-based methods provide an attractive alternative to well-established microbiologic procedures. Mass spectrometry methods can be characterized by the relatively high speed of acquiring taxonomically relevant information. Gel-free mass spectrometry proteomics techniques allow for rapid fingerprinting of bacterial proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or, for high-throughput sequencing of peptides from protease-digested cellular proteins, using mass analysis of fragments from collision-induced dissociation of peptide ions. The latter technique uses database searching of product ion mass spectra. A database contains a comprehensive list of protein sequences translated from protein-encoding open reading frames found in bacterial genomes. The results of such searches allow the assignment of experimental peptide sequences to matching theoretical bacterial proteomes. Phylogenetic profiles of sequenced peptides are then used to create a matrix of sequence-to-bacterium assignments, which are analyzed using numerical taxonomy tools. The results thereof reveal the relatedness between bacteria, and allow the taxonomic position of an investigated strain to be inferred.     

Rapid analysis of tryptically digested cerebrospinal fluid using capillary electrophoresis-electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry

Mass spectrometry has in recent years been established as the method of choice for protein identification and characterization in proteomics. Capillary electrophoresis (CE) is a fast and efficient method for the separation of peptides and proteins. The on-line combination of CE with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) has been shown to be a powerful tool in the analysis of complex mixtures of proteins. This paper presents the first results from a proteomic analysis of human cerebrospinal fluid proteins by tryptic digestion and CE-FTICR-MS, where 30 proteins could be identified on a 95% confidence level with mass measurement errors less than 5 ppm.     

Classification and identification of bacteria using mass spectrometry-based proteomics

Timely classification and identification of bacteria is of vital importance in many areas of public health. Mass spectrometry-based methods provide an attractive alternative to well-established microbiologic procedures. Mass spectrometry methods can be characterized by the relatively high speed of acquiring taxonomically relevant information. Gel-free mass spectrometry proteomics techniques allow for rapid fingerprinting of bacterial proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or, for high-throughput sequencing of peptides from protease-digested cellular proteins, using mass analysis of fragments from collision-induced dissociation of peptide ions. The latter technique uses database searching of product ion mass spectra. A database contains a comprehensive list of protein sequences translated from protein-encoding open reading frames found in bacterial genomes. The results of such searches allow the assignment of experimental peptide sequences to matching theoretical bacterial proteomes. Phylogenetic profiles of sequenced peptides are then used to create a matrix of sequence-to-bacterium assignments, which are analyzed using numerical taxonomy tools. The results thereof reveal the relatedness between bacteria, and allow the taxonomic position of an investigated strain to be inferred.     

Recent advances in protein profiling of tissues and tissue fluids

Creating protein profiles of tissues and tissue fluids, which contain secreted proteins and peptides released from various cells, is critical for biomarker discovery as well as drug and vaccine target selection. It is extremely difficult to obtain pure samples from tissues or tissue fluids, however, and identification of complex protein mixtures is still a challenge for mass spectrometry analysis. Here, we summarize recent advances in techniques for extracting proteins from tissues for mass spectrometry profiling and imaging. We also introduce a novel technique using a capillary ultrafiltration (CUF) probe to enable in vivo collection of proteins from the tissue microenvironment. The CUF probe technique is compared with existing sampling techniques, including perfusion, saline wash, fine-needle aspiration and microdialysis. In this review, we also highlight quantitative mass spectrometric proteomic approaches with, and without, stable-isotope labels. Advances in quantitative proteomics will significantly improve protein profiling of tissue and tissue fluid samples collected by CUF probes.     

Peak intensity prediction in MALDI-TOF mass spectrometry: A machine learning study to support quantitative proteomics

Background  

Mass spectrometry is a key technique in proteomics and can be used to analyze complex samples quickly. One key problem with the mass spectrometric analysis of peptides and proteins, however, is the fact that absolute quantification is severely hampered by the unclear relationship between the observed peak intensity and the peptide concentration in the sample. While there are numerous approaches to circumvent this problem experimentally (e.g. labeling techniques), reliable prediction of the peak intensities from peptide sequences could provide a peptide-specific correction factor. Thus, it would be a valuable tool towards label-free absolute quantification.     

Advances in proteomic workflows for systems biology

Mass spectrometry, specifically the analysis of complex peptide mixtures by liquid chromatography and tandem mass spectrometry (shotgun proteomics) has been at the centre of proteomics research for the past decade. To overcome some of the fundamental limitations of the approach, including its limited sensitivity and high degree of redundancy, new proteomic workflows are being developed. Among these, targeting methods in which specific peptides are selectively isolated, identified and quantified are particularly promising. Here we summarize recent incremental advances in shotgun proteomic methods and outline emerging targeted workflows. The development of the target-driven approaches with their ability to detect and quantify identical, non-redundant sets of proteins in multiple repeat analyses will be crucially important for the application of proteomics to biomarker discovery and validation, and to systems biology research.     

Mass spectrometry detection of monkeypox virus: Comprehensive coverage for ranking the most responsive peptide markers

The recent and sudden outbreak of monkeypox in numerous non-endemic countries requires expanding its surveillance immediately and understanding its origin and spread. As learned from the COVID-19 pandemic, appropriate detection techniques are crucial to achieving such a goal. Mass spectrometry has the advantages of a rapid response, low analytical interferences, better precision, and easier multiplexing to detect various pathogens and their variants. In this proteomic dataset, we report experimental data on the proteome of the monkeypox virus (MPXV) recorded by state-of-the-art shotgun proteomics, including data-dependent and data-independent acquisition for comprehensive coverage. We highlighted 152 viral proteins, corresponding to an overall proteome coverage of 79.5 %. Among the 1371 viral peptides detected, 35 peptides with the most intense signals in mass spectrometry were selected, representing a subset of 13 viral proteins. Their relevance as potential candidate markers for virus detection by targeted mass spectrometry is discussed. This report should assist the rapid development of mass spectrometry-based tests to detect a pathogen of increasing concern.     

Towards the analysis of protein species: an overview about strategies and methods

The deciphering of the relationship between function and exact chemical composition of a defined protein species in the context of the proteome is one of the major challenges in proteomics and molecular cell physiology. In the Special Issue of Amino Acids about the analysis of protein species current approaches are reviewed and new methods described focusing on the investigation of protein species. On the basis of the articles in this Special Issue it can be summarized that first important and promising steps towards the comprehensive analysis of protein species have been done. It is already possible to obtain full (100%) sequence coverage of proteins by mass spectrometry, if the amount of proteins available for their analysis allows their proteolytic degradation by more than one protease and the subsequent mass spectrometric analysis of the resulting peptides. Employing affinity chromatography helps to analyse proteins with defined post-translational modifications thus opening a targeted view on e.g. the phosphoproteome. In the future the aim to identify the exact chemical composition including not one but every posttranslational modification and complete sequence coverage on the protein species level should be achievable with further progress in sample preparation techniques, especially concerning separation techniques on the protein level, mass spectrometry and algorithms for mass spectrometric data processing. For determining the function of defined protein species a closer cooperation between cell biologists and proteomics experts is desirable.     

Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins

A novel gel-free proteomic technology was used to identify more than 800 proteins from 50 million Escherichia coli K12 cells in a single analysis. A peptide mixture is first obtained from a total unfractionated cell lysate, and only the methionine-containing peptides are isolated and identified by mass spectrometry and database searching. The sorting procedure is based on the concept of diagonal chromatography but adapted for highly complex mixtures. Statistical analysis predicts that we have identified more than 40% of the expressed proteome, including soluble and membrane-bound proteins. Next to highly abundant proteins, we also detected low copy number components such as the E. coli lactose operon repressor, illustrating the high dynamic range. The method is about 100 times more sensitive than two-dimensional gel-based methods and is fully automated. The strongest point, however, is the flexibility in the peptide sorting chemistry, which may target the technique toward quantitative proteomics of virtually every class of peptides containing modifiable amino acids, such as phosphopeptides, amino-terminal peptides, etc., adding a new dimension to future proteome research.     

Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine

Mass spectrometric techniques for identification of proteins by "mass fingerprinting" (matching the masses of tryptic peptides from a protein digest to the theoretical peptides in a database) such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) are rapidly growing in popularity as the demand for high throughput analysis of the proteome increases. This is due, in part, to the ability to automate the technique and the rapid rate with which mass spectra may be acquired. An important factor in the accuracy of the technique is the number of tryptic peptides that are identified in the various searching algorithms that exist. The greater sequence coverage of the parent protein that is obtained, the higher the level of confidence in the identification that is determined. One impediment to high levels of sequence coverage is the bias of MALDI-TOF mass spectrometry to arginine-containing peptides. Increasing the sensitivity to lysine-containing peptides should increase the sequence coverage obtained. In order to achieve this result we have developed conditions to modify the epsilon-amine group of lysine in tryptic peptides with O-methylisourea. The conditions utilized result in the conversion of lysine to homoarginine with no modification of the amine terminus of the peptides. The sensitivity of MALDI-TOF mass spectrometry detection of peptides was increased dramatically following modification. The modification chemistry may be applied to tryptic peptide mixtures prior to desalting and spotting onto MALDI-TOF plates. This technique will be particularly useful for identifying proteins with a high lysine/arginine ratio.     

Development and application of proteomics technologies in Saccharomyces cerevisiae

Proteomics research focuses on the identification and quantification of "all" proteins present in cells, organisms or tissue. Proteomics is technically complicated because it encompasses the characterization and functional analysis of all proteins that are expressed by a genome. Moreover, because the expression levels of proteins strongly depend on complex regulatory systems, the proteome is highly dynamic. This review focuses on the two major proteomics methodologies, one based on 2D gel electrophoresis and the other based on liquid chromatography coupled to mass spectrometry. The recent developments of these methodologies and their application to quantitative proteomics are described. The model system Saccharomyces cerevisiae is considered to be the optimal vehicle for proteomics and we review studies investigating yeast adaptation to changes in (nutritional) environment.