Monitoring time‐dependent degradation of phospholipids in sectioned tissues by MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) is useful for visualizing the localization of phospholipids on biological tissue surfaces creating great opportunities for IMS in lipidomic investigations. With advancements in IMS of lipids, there is a demand for large‐scale tissue studies necessitating stable, efficient and well‐defined sample handling procedures. Our work within this article shows the effects of different storage conditions on the phospholipid composition of sectioned tissues from mouse organs. We have taken serial sections from mouse brain, kidney and liver thaw mounted unto ITO‐coated glass slides and stored them under various conditions later analyzing them at fixed time points. A global decrease in phospholipid signal intensity is shown to occur and to be a function of time and temperature. Contrary to the global decrease, oxidized phospholipid and lysophospholipid species are found to increase within 2 h and 24 h, respectively, when mounted sections are kept at ambient room conditions. Imaging experiments reveal that degradation products increase globally across the tissue. Degradation is shown to be inhibited by cold temperatures, with sample integrity maintained up to a week after storage in ?80 °C freezer under N₂ atmosphere. Overall, the results demonstrate a timeline of the effects of lipid degradation specific to sectioned tissues and provide several lipid species which can serve as markers of degradation. Importantly, the timeline demonstrates oxidative sample degradation begins appearing within the normal timescale of IMS sample preparation of lipids (i.e. 1–2 h) and that long‐term degradation is global. Taken together, these results strengthen the notion that standardized procedures are required for phospholipid IMS of large sample sets, or in studies where many serial sections are prepared together but analyzed over time such as in 3‐D IMS reconstruction experiments. Copyright © 2014 John Wiley & Sons, Ltd.     

High‐speed MALDI MS/MS imaging mass spectrometry using continuous raster sampling

A matrix‐assisted laser desorption/ionization time of flight/time of flight tandem mass spectrometer (MALDI TOF/TOF) has been used for high‐speed precursor/fragment ion transition image acquisition. High‐throughput analysis is facilitated by an Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz, a high digitizer acquisition rate (up to 50 pixels/s), and continuous laser raster sampling. MS/MS experiments are enabled through the use of a precision timed ion selector, second source acceleration, and a dedicated collision cell. Continuous raster sampling is shown here to facilitate rapid MS/MS ion image acquisition from thin tissue sections for the drug rifampicin and for a common kidney lipid, SM4s(d18:1/24:1). The ability to confirm the structural identity of an analyte as part of the MS/MS imaging experiment is an essential part of the analysis. Additionally, the increase in sensitivity and specificity afforded by an MS/MS approach is highly advantageous, especially when interrogating complex chemical environments such as those in biological tissues. Herein, we report continuous laser raster sampling TOF/TOF imaging methodologies which demonstrate 8 to 14‐fold increases in throughput compared with existing MS/MS instrumentation, an important advantage when imaging large areas on tissues. Copyright © 2015 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.     

Uncovering matrix effects on lipid analyses in MALDI imaging mass spectrometry experiments

The specific matrix used in matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT‐ICR) IMS of murine liver tissue when using 9‐aminoacridine (9AA), 5‐chloro‐2‐mercaptobenzothiazole (CMBT), 1,5‐diaminonaphthalene (DAN), 2,5‐Dihydroxyacetophenone (DHA), and 2,5‐dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.     

MALDI imaging mass spectrometry reveals multiple clinically relevant masses in colorectal cancer using large‐scale tissue microarrays

For identification of clinically relevant masses to predict status, grade, relapse and prognosis of colorectal cancer, we applied Matrix‐assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) to a tissue micro array containing formalin‐fixed and paraffin‐embedded tissue samples from 349 patients. Analysis of our MALDI‐IMS data revealed 27 different m/z signals associated with epithelial structures. Comparison of these signals showed significant association with status, grade and Ki‐67 labeling index. Fifteen out of 27 IMS signals revealed a significant association with survival. For seven signals (m/z 654, 776, 788, 904, 944, 975 and 1013) the absence and for eight signals (m/z 643, 678, 836, 886, 898, 1095, 1459 and 1477) the presence were associated with decreased life expectancy, including five masses (m/z 788, 836, 904, 944 and 1013) that provided prognostic information independently from the established prognosticators pT and pN. Combination of these five masses resulted in a three‐step classifier that provided prognostic information superior to univariate analysis. In addition, a total of 19 masses were associated with tumor stage, grade, metastasis and cell proliferation. Our data demonstrate the suitability of combining IMS and large‐scale tissue micro arrays to simultaneously identify and validate clinically useful molecular marker. Copyright © 2017 John Wiley & Sons, Ltd.     

Novel vacuum stable ketone‐based matrices for high spatial resolution MALDI imaging mass spectrometry

We describe the use of aromatic ketones and cinnamyl ketones that have high vacuum stability for analyzing tissue sections using matrix‐assisted laser desorption/ionization imaging mass spectrometry. Specifically, the matrix, (E)‐4‐(2,5‐dihydroxyphenyl)but‐3‐en‐2‐one (2,5‐cDHA) provides high sensitivity and high vacuum stability while producing small size crystals (1‐2 μm). A high throughput and highly reproducible sample preparation method was developed for these matrices that first involves using an organic spray solution for small matrix crystal seeding followed by spraying of the matrix in a 30% acetonitrile/70% water solution on the tissue surface to obtain a homogeneous coating of small crystals, suitable for high spatial resolution imaging.     

MALDI‐MS profiling of serum O‐glycosylation and N‐glycosylation in COG5‐CDG

Congenital disorders of glycosylation (CDG) are due to defective glycosylation of glycoconjugates. Conserved oligomeric Golgi (COG)‐CDG are genetic diseases due to defects of the COG complex subunits 1–8 causing N‐glycan and O‐glycan processing abnormalities. In COG‐CDG, isoelectric focusing separation of undersialylated glycoforms of serum transferrin and apolipoprotein C‐III (apoC‐III) allows to detect N‐glycosylation and O‐glycosylation defects, respectively. COG5‐CDG (COG5 subunit deficiency) is a multisystem disease with dysmorphic features, intellectual disability of variable degree, seizures, acquired microcephaly, sensory defects and autistic behavior. We applied matrix‐assisted laser desorption/ionization‐MS for a high‐throughput screening of differential serum O‐glycoform and N‐ glycoform in five patients with COG5‐CDG. When compared with age‐matched controls, COG5‐CDG showed a significant increase of apoC‐III_0a (aglycosylated glycoform), whereas apoC‐III₁ (mono‐sialylated glycoform) decreased significantly. Serum N‐glycome of COG5‐CDG patients was characterized by the relative abundance of undersialylated and undergalactosylated biantennary and triantennary glycans as well as slight increase of high‐mannose structures and hybrid glycans. Using advanced and well‐established MS‐based approaches, the present findings reveal novel aspects on O‐glycan and N‐glycan profiling in COG5‐CDG patients, thus providing an increase of current knowledge on glycosylation defects caused by impairment of COG subunits, in support of clinical diagnosis. Copyright © 2017 John Wiley & Sons, Ltd.     

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.     

Sucrose cryo‐protection facilitates imaging of whole eye sections by MALDI mass spectrometry

Sucrose is used as a cryo‐preservation agent on large mammalian eyes post formalin fixation and is shown to reduce freezing artefacts allowing the collection of 12‐µm thick sections from these large aqueous samples. The suitability of this technique for use in MALDI imaging experiments is demonstrated by the acquisition of the first images of lipid distributions within whole sagittal porcine eye sections. Copyright © 2012 John Wiley & Sons, Ltd.     

MALDI imaging mass spectrometry of β‐ and γ‐crystallins in the ocular lens

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age‐related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age‐related modifications. Previous work demonstrated that, under standard protein IMS conditions, α‐crystallin signals dominated the mass spectrum and age‐related modifications to α‐crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of β‐ and γ‐crystallins in ocular lens tissue. Acquired images showed that γ‐crystallins were localized predominately in the lens nucleus whereas β‐crystallins were primarily localized to the lens cortex. Age‐related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top‐down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC‐MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.     

Orthogonal organic and aqueous‐based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous‐based buffer for tissue section preparation before matrix‐assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water–acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14‐day mouse fetus whole‐body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on‐tissue MALDI analysis compared with solely conventional organic rinsing. Copyright © 2013 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.     

Allergenic compounds on the inner and outer surfaces of natural latex gloves: MALDI mass spectrometry and imaging of proteinous allergens

Natural latex gloves are the cause of a severe health problem to an increasing number of healthcare workers or patients due to the presence of protein allergens as Hevein or Rubber Elongation Factor (REF). One of the most challenging problems is the in situ localization of theses allergens in, e.g. gloves, to estimate the allergenic potential of the latex material. A sample preparation protocol applying a binary matrix-assisted laser desorption/ionization(MALDI) matrix containing alpha-cyano-4-hydroxy cinnamic acid (CHCA) and 2,5-dihydroxy benzoic acid (DHB) on trifluoro acetic acid (TFA) etched latex glove surfaces allowed the direct determination (exact molecular weight) of Hevein, REF and a truncated form of REF (tREF) within nine different brands of natural latex gloves by means of MALDI-TOF-MS in the linear mode. MALDI mass spectrometry demonstrated that Hevein, tREF and REF were present on the inner surfaces (in direct contact with the skin) of many, but not all, investigated gloves without any prior extraction procedure. Additionally, different isoforms of the allergen Hevein were detected (exhibiting ragged C-termini). tREF and REF could always be detected beside each other, but were not observed on every latex glove sample, which contained Hevein. It was also demonstrated that there is a significant difference in terms of proteins and polymers between inner and outer surfaces of gloves, which helps to explain the different allergenic potential of these.MALDI imaging allowed for the first time the unambiguous localization of all three allergens in parallel and showed that Hevein was present on 36% of the investigated area of a latex glove with a certain localization, whereupon, tREF and REF were only found on 25% of the investigated material.     

Fragmentation of negative ions from N‐linked carbohydrates,Part 4. Fragmentation of complex glycans lacking substitution on the 6‐antenna

Negative ion CID spectra of N‐linked glycans released from glycoproteins contain many ions that are diagnostic for specific structural features such as the detailed arrangement of antennae and the location of fucose residues. Identification of such ions requires reference glycans that are often difficult to acquire in a pure state. The recent acquisition of a sample of N‐glycans from a patient lacking the enzyme N‐acetylglucosaminyltransferase‐2 provided an opportunity to investigate fragmentation of glycans lacking a 6‐antenna. These glycans contained one or two galactose‐N‐acetylglucosamine‐chains attached to the 3‐linked mannose residue of the trimannosyl‐chitobiose core with and without fucose substitution. The spectra from the patient sample clearly defined the antenna distribution and showed striking differences from the spectra of isomeric compounds obtained from normal subjects. Furthermore, they provided additional information on previously identified antenna‐specific fragment ions and indicated the presence of additional ions that were diagnostic of fucose substitution. Glycans obtained from such enzyme‐deficient patients can, thus, be a valuable way of obtaining spectra of specific isomers in a relatively pure state for interpretation of mass spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

N‐ and O‐linked glycosylation site profiling of the human basic salivary proline‐rich protein 3M

In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline‐rich protein 3M, encoded by PRB3‐M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed‐phase high‐performance liquid chromatography with high‐resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N‐deglycosylation with Peptide‐N‐Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N‐ and O‐glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O‐linked to Threonine 50, and 33 different glycans N‐linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.     

Identification of N-linked glycosylation sites in human nephrin using mass spectrometry

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.     

Simultaneous detection of phosphatidylcholines and glycerolipids using matrix‐enhanced surface‐assisted laser desorption/ionization‐mass spectrometry with sputter‐deposited platinum film

Matrix‐assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) allows for the simultaneous detection and imaging of several molecules in brain tissue. However, the detection of glycerolipids such as diacylglycerol (DAG) and triacylglycerol (TAG) in brain tissues is hindered in MALDI‐IMS because of the ion suppression effect from excessive ion yields of phosphatidylcholine (PC). In this study, we describe an approach that employs a homogeneously deposited metal nanoparticle layer (or film) for the detection of glycerolipids in rat brain tissue sections using IMS. Surface‐assisted laser desorption/ionisation IMS with sputter‐deposited Pt film (Pt‐SALDI‐IMS) for lipid analysis was performed as a solvent‐free and organic matrix‐free method. Pt‐SALDI produced a homogenous layer of nanoparticles over the surface of the rat brain tissue section. Highly selective detection of lipids was possible by MALDI‐IMS and Pt‐SALDI‐IMS; MALDI‐IMS detected the dominant ion peak of PC in the tissue section, and there were no ion peaks representing glycerolipids such as DAG and TAG. In contrast, Pt‐SALDI‐IMS allowed the detection of these glycerolipids, but not PC. Therefore, using a hybrid method combining MALDI and Pt‐SALDI (i.e., matrix‐enhanced [ME]‐Pt‐SALDI‐IMS), we achieved the simultaneous detection of PC, PE and DAG in rat brain tissue sections, and the sensitivity for the detection of these molecules was better than that of MALDI‐IMS or Pt‐SALDI alone. The present simple ME‐Pt‐SALDI approach for the simultaneous detection of PC and DAG using two matrices (sputter‐deposited Pt film and DHB matrix) would be useful in imaging analyses of biological tissue sections. Copyright © 2015 John Wiley & Sons, Ltd.     

Low C24‐OH and C22‐OH sulfatides in human renal cell carcinoma

Histopathologic diagnosis of renal cell carcinoma (RCC) may sometimes be difficult with small biopsy samples. We applied histology‐directed matrix‐assisted laser desorption/ionization mass spectrometry to RCC samples to evaluate whether and how lipid profiles are different between RCC and normal tissue. We evaluated 59 RCC samples and 24 adjacent normal tissue samples collected from patients who underwent surgery. Five peaks were significantly differently expressed (p < 10^?7) between RCCs and adjacent normal tissue samples. C24‐OH sulfatide (ST‐OH {18:1/24:0}[M‐H]^?; m/z 906.7 in the negative ion mode) and C22‐OH sulfatide (ST‐OH {18:1/22:0}[M‐H]^?; m/z 878.6 in the negative ion mode) were most significantly underexpressed in RCC samples, compared with adjacent normal tissue samples. With 100 random training‐to‐test partitions within these samples, the median prediction accuracy (RCC vs. normal) ranged from 96.3% to 100% at p cutoff values for feature selection ranging from 0.001 to 10^?7. Two oncocytoma samples were predicted as normal tissue by five lipids that were differentially expressed between RCC and normal tissue at p < 10^?7. Clear‐cell, papillary, and chromophobe RCCs were different in lipid profiles. Permutation p‐ values for 0.632+ bootstrap cross‐validated misclassification rates were less than 0.05 for all the classifiers. Thus, lipid profiles differentiate RCC from normal tissue and may possibly classify the histology of RCC. © 2014 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons, Ltd.     

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.     

Travelling‐wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N‐glycans

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1‐acid glycoprotein and thyroglobulin were measured with a travelling‐wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man₃GlcNAc₃ from chicken ovalbumin and Man₃GlcNAc₃Fuc₁ from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision‐induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high‐mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high‐mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility‐m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.