Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.
19 Related JoVE Articles!
Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water
Institutions: Boston Biomedical Research Institute.
Stable isotopes are essential tools in biological mass spectrometry. Historically, 18
O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3
. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18
O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (
reviewed by Fenselau and Yao4
, Miyagi and Rao5
and Ye et al.6)
O-labeling constitutes a simple and low-cost alternative to chemical (e.g.
iTRAQ, ICAT) and metabolic (e.g.
SILAC) labeling techniques7
. Depending on the protease utilized, 18
O-labeling can result in the incorporation of up to two 18
O-atoms in the C-terminal carboxyl group of the cleavage product3
. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8
. In our PALeO (p
-enriched water) adaptation of enzymatic 18
O-labeling, we utilized 50% 18
O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9
and monitor proteolytic reactions10-11
. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18
O-atom. Such "double-labeling" enzymes can be used for postdigestion 18
O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18
O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18
O-stable isotope signatures into peptides.
Biochemistry, Issue 72, Molecular Biology, Proteins, Proteomics, Chemistry, Physics, MALDI-TOF mass spectrometry, proteomics, proteolysis, quantification, stable isotope labeling, labeling, catalyst, peptides, 18-O enriched water
Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation
Institutions: Aix Marseille Université, Hôpital de la Timone.
MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.
Immunology, Issue 82, MALDI-TOF, mass spectrometry, fingerprint, Macrophages, activation, IFN-g, TNF, LPS, IL-4, bacterial pathogens
Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure
Institutions: George Washington University.
Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1
The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 μm wavelength utilize the sudden excitation of water that results in phase explosion.2
Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11
In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12
This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa
bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.
Cellular Biology, Issue 43, single cell analysis, mass spectrometry, laser ablation electrospray ionization, LAESI, metabolomics, direct analysis
Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry
Institutions: Bielefeld University.
This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g.
, dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.
Chemistry, Issue 91, mass spectrometry, electron impact, inlet system, volatile, air sensitive
Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation
Institutions: Tunghai University, University of California, Riverside .
Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3
. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4
. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2
, instead of a HPLC/ESI (electrospray ionization)-MS system5,6
. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called γ-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2
). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9
Biochemistry, Issue 57, Deuterium, H/D exchange, Mass Spectrometry, Pak2, Caspase 3, MALDI-TOF
Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry
Institutions: Westmead Hospital, Westmead Hospital, Westmead Hospital.
An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.
Immunology, Issue 87, Gram negative bacilli, blood culture, blood stream infection, bacteraemia, MALDI-TOF, mass spectrometry
Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome
Institutions: University of South Florida.
Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.
Biochemistry, Issue 85, Cochlear, chromatography, LC-MS/MS, mass spectrometry, Proteomics, sensory epithelium
A Strategy for Sensitive, Large Scale Quantitative Metabolomics
Institutions: Cornell University, Cornell University.
Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously. Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.
Chemistry, Issue 87, high-resolution mass spectrometry, metabolomics, positive/negative switching, low mass calibration, Orbitrap
OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides
Institutions: University of California, Berkeley, University of California, Berkeley.
The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1
(Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2
, and is amenable to high-throughput analyses3
. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4
. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13
, xylan using an endo
, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8
. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9
Plant Biology, Issue 40, Extracellular matrix, cell walls, polysaccharides, glycosylhydrolase, MALDI-TOF mass spectrometry
The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics
), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP
in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Institutions: The University of Texas at Austin, The University of Texas at Austin, The University of Texas at Austin.
Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.
Chemistry, Issue 79, Membrane Lipids, Toll-Like Receptors, Endotoxins, Glycolipids, Lipopolysaccharides, Lipid A, Microbiology, Lipids, lipid A, Bligh-Dyer, thin layer chromatography (TLC), lipopolysaccharide, mass spectrometry, Collision Induced Dissociation (CID), Photodissociation (PD)
Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing
Institutions: University Hospital Center and University of Lausanne.
Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.
Immunology, Issue 92, blood culture, bacteriology, identification, antibiotic susceptibility testing, MALDI-TOF MS.
Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry
Institutions: Georgia Institute of Technology.
Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo
. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.
Bioengineering, Issue 77, Molecular Biology, Biomedical Engineering, Chemistry, Biochemistry, Biophysics, Physics, Cellular Biology, Molecular Imaging, Mass Spectrometry, MS, MSI, Desorption electrospray ionization, DESI, Ambient mass spectrometry, tissue, sectioning, biomarker, imaging
MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules
Institutions: University of Wisconsin- Madison, University of Wisconsin- Madison.
Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1
. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5
. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.
Basic Protocol, Issue 85, Mass Spectrometric Imaging, Imaging Mass Spectrometry, MALDI, TOF/TOF, Medicago truncatula, Metabolite, Small Molecule, Sublimation, Automatic Sprayer
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
Institutions: University of Victoria, University of Victoria.
Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.
Basic Protocol, Issue 81, eye, molecular imaging, chemistry technique, analytical, mass spectrometry, matrix assisted laser desorption/ionization (MALDI), tandem mass spectrometry, lipid, tissue imaging, bovine lens, dithranol, matrix, FTICR (Fourier Transform Ion Cyclotron Resonance)
Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery
Institutions: Protein Discovery, Inc..
The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free
of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1
Basic Protocols, Cellular Biology, Issue 34, GELFREE, SDS PAGE, gel electrophoresis, protein fractionation, separation, electrophoresis, proteomics, mass spectrometry
MALDI Sample Preparation: the Ultra Thin Layer Method
Institutions: Rockefeller University.
This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2
. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g.
dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2
. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3
. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.
Cellular Biology, Issue 3, mass-spectrometry, ultra-thin layer, MALDI, MS, proteins