A recommended and verified procedure for in situ tryptic digestion of formalin‐fixed paraffin‐embedded tissues for analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin‐fixed paraffin‐embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross‐links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.     

Rapid identification of pesticides in human oral fluid for emergency management by thermal desorption electrospray ionization/mass spectrometry

Self‐poisoning with pesticides accounts for approximately one‐third of all suicides worldwide. To expedite rescue in the emergency department, it is essential to develop a point‐of‐care analytical method for rapid identification of ingested pesticides. In this study, five of the most common pesticides ingested by self‐poisoning patients in Taiwan were analyzed from oral fluid samples. Pesticide‐oral fluid mixtures were applied on a cotton swab and then transferred into methanol. A metallic probe was used to sample the methanol solution for subsequent thermal desorption‐electrospray ionization mass spectrometry analysis. Altogether, pesticide sampling, transfer, desorption, ionization, and detection took less than 1 min. The reproducibility of this method (n = 6) was shown in the observed low‐relative standard deviation (<7%) in the detection of pesticide in oral fluid. The detection limits of the pesticides in oral fluid obtained from four human subjects by thermal desorption‐electrospray ionization mass spectrometry were between 1–10 ppb with relative standard deviation 10.7%. Moreover, in this study, linear responses of five pesticides in oral fluid with concentrations between 1 ppb–1 ppm (R2 between 0.9938 and 0.9988) were observed. As the whole analytical process is extremely short, this technique allows for early non‐invasive point‐of‐care identification of pesticides in the oral fluid of self‐poisoning patients in the emergency room, providing important toxicological information for decision‐making during critical resuscitation. Copyright © 2016 John Wiley & Sons, Ltd.     

Visualization of cancer‐related chemical components in mouse pancreas tissue by tapping‐mode scanning probe electrospray ionization mass spectrometry

Mass spectrometry imaging is an informative approach for the comprehensive analysis of multiple components inside biological specimens. We used novel tapping‐mode scanning probe electrospray ionization mass spectrometry method to visualize cancer‐related chemical components in the mouse pancreas tissue section at a sampling pitch of 100 µm. Positive ion mode measurements from m/z 100 to 1500 resulted in the visualization of multiple components that are tentatively assigned as polyamines, lipids and proteins. Their signal intensities inside the cancerous and the non‐cancerous regions were found to be significantly different by the two‐sample t‐test. Copyright © 2015 John Wiley & Sons, Ltd.     

A technique for relative quantitation of cancer biomarkers in formalin‐fixed,paraffin‐embedded (FFPE) tissue using stable‐isotope‐label based mass spectrometry imaging (SILMSI)

We developed a novel technique for the relative quantitation of pairs of cancer biomarkers in formalin‐fixed paraffin‐embedded (FFPE) tissue. The method utilizes stable isotope labeled (SIL) chromogens deposited during the standard immunohistochemistry (IHC) tissue staining process. The labeled chromogens are precipitated on tissue enzymatically using the standard IHC protocols. The tissue is then imaged with matrix‐free laser desorption ionization time‐of‐flight mass spectrometry, and peak intensities of reporter ions are used to estimate the relative quantitation of protein biomarkers across the tissue. The relative abundance of two breast cancer biomarkers, estrogen receptor (ER) and progesterone receptor (PgR), were quantitated using their ratio of expression in xenograft models, and the ratios were found to be reproducible both within and across serial sections. The relative quantification of multiple biomarkers in situ across a single tissue section adds an additional dimension in cancer histological evaluation by allowing a visual and statistical assessment of tumor heterogeneity. Copyright © 2015 John Wiley & Sons, Ltd.     

Protein analysis by desorption electrospray ionization mass spectrometry and related methods

Desorption electrospray ionization mass spectrometry (DESI‐MS) requires little to no sample preparation and has been successfully applied to the study of biologically significant macromolecules such as proteins. However, DESI‐MS and other ambient methods that use spray desorption to process samples during ionization appear limited to smaller proteins with molecular masses of 25 kDa or less, and a decreasing instrumental response with increasing protein size has often been reported. It has been proposed that this limit results from the inability of some proteins to easily desorb from the surface during DESI sampling. The present study investigates the apparent mass dependence of the instrumental response observed during the DESI‐MS analysis of proteins using spray desorption collection and reflective electrospray ionization. Proteins, as large as 66 kDa, are shown to be quantitatively removed from surfaces by using spray desorption collection. However, incomplete dissolution and the formation of protein–protein and protein–contaminant clusters appear to be responsible for the mass‐dependent loss in sensitivity for protein analysis. Alternative ambient mass spectrometry approaches that address some of the problems encountered by spray desorption techniques for protein analysis are also discussed. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular histology analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry using gold nanoparticles as matrix

Gold nanoparticles (AuNPs) were applied and optimized as matrix for matrix‐assisted laser desorption/ionization mass spectrometry analysis of animal tissues, and enabled histological analysis of animal tissues at molecular level by imaging mass spectrometry (IMS). AuNPs were coated on animal tissue in a solvent‐free manner via argon ion sputtering. Metabolites, including neurotransmitters, fatty acids and nucleobases, were directly detected from mouse brain tissue. Based on region‐specific chemical profiles, fine histological features of mouse brain tissue and heterogeneous regions of tumor tissue were both revealed. Copyright © 2011 John Wiley & Sons, Ltd.     

A Comparison of Tissue Spray and Lipid Extract Direct Injection Electrospray Ionization Mass Spectrometry for the Differentiation of Eutopic and Ectopic Endometrial Tissues

Recent research revealed that tissue spray mass spectrometry enables rapid molecular profiling of biological tissues, which is of great importance for the search of disease biomarkers as well as for online surgery control. However, the payback for the high speed of analysis in tissue spray analysis is the generally lower chemical sensitivity compared with the traditional approach based on the offline chemical extraction and electrospray ionization mass spectrometry detection. In this study, high resolution mass spectrometry analysis of endometrium tissues of different localizations obtained using direct tissue spray mass spectrometry in positive ion mode is compared with the results of electrospray ionization analysis of lipid extracts. Identified features in both cases belong to three lipid classes: phosphatidylcholines, phosphoethanolamines, and sphingomyelins. Lipids coverage is validated by hydrophilic interaction liquid chromatography with mass spectrometry of lipid extracts. Multivariate analysis of data from both methods reveals satisfactory differentiation of eutopic and ectopic endometrium tissues. Overall, our results indicate that the chemical information provided by tissue spray ionization is sufficient to allow differentiation of endometrial tissues by localization with similar reliability but higher speed than in the traditional approach relying on offline extraction.

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Self‐Assembly of Aniline Oligomers

A great number of nano/microscaled morphologies have recently been prepared during the oxidation of aniline. At the early stage of oxidation, aniline oligomers are obtained, often in spectacular morphologies depending on reaction conditions. Herein, the flower‐like hierarchical architectures assembled from aniline oligomers by a template‐free method are reported. Their formation process is ascribed to the self‐assembly of oligoanilines through non‐covalent interactions, such as hydrogen bonding, hydrophobic forces, and π–π stacking. The model of directional growth is offered to explain the formation of petal‐like objects and, subsequently, flowers. In order to investigate the chemical structure of the oligomers, a series of characterizations have been carried out, such as matrix‐assisted laser desorption ionization, time‐of‐flight mass spectrometry, gas chromatography coupled with mass spectrometry analysis, X‐ray diffraction, and UV/Vis, Fourier‐transform infrared, and Raman spectroscopies. Based on the results of characterization methods, a formation mechanism for aniline oligomers and their self‐assembly is proposed.     

Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization and application of a self‐aspirating electrospray source with pneumatic‐assisted ionization

A single gas‐assisted electrospray ion source developed for ambient mass spectrometry is introduced in this paper. Simultaneous self‐aspiration and electrospray could be achieved by using a constant sheath gas flow supplied from a mini air pump. A gas dynamic study of the spray module is carried out for structural optimization. The entire device exhibits a simplified design and has been systematically characterized through both simulated and experimental investigations. According to the results, the ion source exhibited satisfactory stability and the ability for quantitative operation in routine electrospray ionization mass spectrometry. Furthermore, the ion source can be operated as a desorption electrospray ionization source to perform direct desorption/ionization of the solid samples. The versatile source described here appears to provide a practical approach to perform ambient mass spectrometry analysis with unrestricted sampling operation, and the extensive gas dynamic studies together with the experimental characterization are believed to be helpful in building self‐aspirating spray devices. Copyright © 2017 John Wiley & Sons, Ltd.     

Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

A derivatization and validation strategy for determining the spatial localization of endogenous amine metabolites in tissues using MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here, we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix‐assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4‐hydroxy‐3‐methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high‐mass resolution and MSⁿ IMS. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high‐performance liquid chromatography (HPLC)‐MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds. Copyright © 2014 John Wiley & Sons, Ltd.     

Extracellular matrix alterations in low‐grade lung adenocarcinoma compared with normal lung tissue by imaging mass spectrometry

Lung adenocarcinoma (LUAD) is the second most common cancer, affecting both men and women. Fibrosis is a hallmark of LUAD occurring throughout progression with excess production of extracellular matrix (ECM) components that lead to metastatic cell processes. Understanding the ECM cues that drive LUAD progression has been limited due to a lack of tools that can access and report on ECM components within the complex tumor microenvironment. Here, we test whether low‐grade LUAD can be distinguished from normal lung tissue using a novel ECM imaging mass spectrometry (ECM IMS) approach. ECM IMS analysis of a tissue microarray with 20 low‐grade LUAD tissues and 20 normal lung samples from 10 patients revealed 25 peptides that could discriminate between normal and low‐grade LUAD using area under the receiver‐operating curve (AUC) ≥0.7, P value ≤.001. Principal component analysis demonstrated that 62.4% of the variance could be explained by sample origin from normal or low‐grade tumor tissue. Additional work performed on a wedge resection with moderately differentiated LUAD demonstrated that the ECM IMS analytical approach could distinguish LUAD spectral features from spectral features of normal adjacent lung tissue. Conventional liquid chromatography with tandem mass spectrometry (LC‐MS/MS) proteomics demonstrated that specific sites of hydroxylation of proline (HYP) were a main collagen post translational modification that was readily detected in LUAD. A distinct peptide from collagen 3A1 modified by HYP was increased 3.5 fold in low‐grade LUAD compared with normal lung tissue (AUC 0.914, P value <.001). This suggests that regulation of collagen proline hydroxylation could be an important process during early LUAD fibrotic deposition. ECM IMS is a useful tool that may be used to define fibrotic deposition in low‐grade LUAD.     

Tandem Mass Spectrometric Characterization of Fetuin Sialylated Glycopeptides Enriched by TiO2 Microcolumn

Sialylation of glycoproteins is vital for the function or physicochemical properties of a protein. It becomes more and more important to develop approaches that can be used to efficiently isolate and identify sialylated glycopeptides or glycoproteins for monitoring changes in glycoproteome. In the present study, we analyze intact structures of the enriched sialylated glycopeptides of bovine fetuin by matrix‐assisted laser desorption/ionization‐tandem mass spectrometry (MALDI‐MS/MS), without any chemical derivation. The experimental data show that the optimal loading buffer for TiO₂ as matrix is 80% acetonitrile/2% TFA (trifluoroacetic acid)/100 mg/mL DHB (2,5‐dihydroxybenzoic acid) which is also compatible with MALDI‐mass spectrometric analysis. This study indicates that the improved enrichment approach combined with MALDI‐MS/MS may be a powerful tool to analyze intact structures and components of the sialylated glycopeptides from complex peptide mixture.     

Design of Experiment (DoE) as a Tool for the Optimization of Source Conditions in SEC‐ESI‐MS of Functional Synthetic Polymers Synthesized via ATRP

Design of experiment (DoE) is applied to establish the optimum ionization conditions for analyzing synthetic polymers via coupled size exclusion chromatography electrospray ionization mass spectrometry (SEC‐ESI‐MS) yielding maximum ionization efficiency. The ion source conditions were optimized with regard to the ionization efficiency, the amount of fragmentation, as well as the formation of salt adducts. A D ‐optimal experimental design was employed for this purpose and the recorded data were evaluated by a quadratic response surface model, accounting for possible interactions between the individual source settings. It was established that the ionization efficiency can be improved by up to one order of magnitude without compromising the softness of the ionization process and that optimal ionization conditions are found at similar source settings regardless of the charge state. The present optimization exercise therefore provides a hands‐on guide for the use of experimental design to determine optimum ionization conditions during the SEC‐ESI‐MS of functional polymers.

     

Displaced dual‐mode imaging with desorption electrospray ionization for simultaneous mass spectrometry imaging in both polarities and with several scan modes

Displaced dual‐mode imaging (DDI) is introduced as a method for simultaneous imaging in positive and negative‐ion mode on the same sample with desorption electrospray ionization imaging, as well as a method for simultaneous imaging in full‐scan and tandem mass spectrometry (MS/MS) mode. DDI is performed by using a smaller row distance in the y‐direction than the desired image resolution and recording for example every second row in positive‐ion mode and the other half of the rows in negative‐ion mode, thus resulting in two separate images. This causes some degree of oversampling, which is thus utilized to obtain complementary mass spectrometric of the sample. Imaging with both polarities is exemplified on an imprint of a Hypericum perforatum leaf containing secondary metabolites which ionize in both polarites and a mouse kidney containing phospholipids which ionize in positive or negative mode only. Simultaneous full‐scan and MS/MS imaging was demonstrated on the same mouse kidney, as the mouse had been given a relatively low dose of the antidepressive drug amitriptyline. While the full‐scan data allowed imaging of the endogenous phospholipids, the drug and its metabolites were only visible in the MS/MS images. The latter approach is useful, for example in whole‐body imaging experiments where the full‐scan data gives an overview of the tissue, and the MS/MS mode provides the sensitivity to image trace amounts of drugs and metabolites. Copyright © 2013 John Wiley & Sons, Ltd.     

MALDI‐FT‐ICR‐MS for archaeological lipid residue analysis

Soft‐ionization methods are currently at the forefront of developing novel methods for analysing degraded archaeological organic residues. Here, we present little‐used soft ionization method of matrix assisted laser desorption/ionization‐Fourier transform‐ion cyclotron resonance‐mass spectrometry (MALDI‐FT‐ICR‐MS) for the identification of archaeological lipid residues. It is a high‐resolution and sensitive method with low limits of detection capable of identifying lipid compounds in small concentrations, thus providing a highly potential new technique for the analysis of degraded lipid components. A thorough methodology development for analysing cooked and degraded food remains from ceramic vessels was carried out, and the most efficient sample preparation protocol is described. The identified components, also controlled by independent parallel analysis by gas chromatography‐mass spectrometry (GC‐MS) and gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS), demonstrate its capability of identifying very different food residues including dairy, adipose fats as well as lipids of aquatic origin. The results obtained from experimentally cooked and original archaeological samples prove the suitability of MALDI‐FT‐ICR‐MS for analysing archaeological organic residues. Sample preparation protocol and identification of compounds provide future reference for analysing various aged and degraded lipid residues in different organic and mineral matrices.     

An approach for visualizing the spatial metabolome of an entire plant root system inspired by the Swiss‐rolling technique

The spatial configuration and morphology of roots are commonly monitored for a better understanding of plant health and development. However, this approach provides minimal details about the biochemistry regulating the observable traits. Therefore, the ability to metabolically map the entire root structure would be of major value. Here, we developed a sample preparation approach that enables imaging of the entire root within a restricted space (width of microscope slide), which was influenced by the Swiss‐rolling technique. We were able to image and confidently identify molecules along the entire root structure from rolled‐root tissue sections using multiple spatially resolved mass spectrometry approaches.     

Study of the noncovalent interactions of ginsenosides and amyloid‐β‐peptide by CSI‐MS and molecular docking

Noncovalent interactions between drugs and proteins play significant roles for drug metabolisms and drug discoveries. Mass spectrometry has been a commonly used method for studying noncovalent interactions. However, the harsh ionization process in electrospray ionization mass spectrometry (ESI‐MS) is not conducive to the preservation of noncovalent and unstable biomolecular complexes compared with the cold spray ionization mass spectrometry (CSI‐MS). A cold spray ionization providing a stable solvation‐ionization at low temperature is milder than ESI, which was more suitable for studying noncovalent drug‐protein complexes with exact stoichiometries. In this paper, we apply CSI‐MS to explore the interactions of ginsenosides toward amyloid‐β‐peptide (Aβ) and clarify the therapeutic effect of ginsenosides on Alzheimer's disease (AD) at the molecular level for the first time. The interactions of ginsenosides with Aβ were performed by CSI‐MS and ESI‐MS, respectively. The ginsenosides Rg1 bounded to Aβ at the stoichiometries of 1:1 to 5:1 could be characterized by CSI‐MS, while dehydration products are more readily available by ESI‐MS. The binding force depends on the number of glycosyls and the type of ginsenosides. The relative binding affinities were sorted in order as follows: Rg1 ≈ Re > Rd ≈ Rg2 > Rh2, protopanaxatriol by competition experiments, which were supported by molecular docking experiment. CSI‐MS is expected to be a more appropriate approach to determine the weak but specific interactions of proteins with other natural products especially polyhydroxy compounds.     

Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC‐MS/MS

A liquid chromatography electrospray ionization tandem mass spectrometry (LC‐MS/MS) method has been developed for the determination of 5,10‐methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5‐methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC‐MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut‐off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC‐MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right‐ and left‐sided tumors follows different pathways. Copyright © 2012 John Wiley & Sons, Ltd.