Enhancement of Tryptic Peptide Signals from Tissue Sections Using MALDI IMS Postionization (MALDI-2)

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization MALDI-2 has been shown to increase sensitivity for several molecular classes but thus far this has not been reported for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (termed MALDI-1), and conventional MALDI augmented using a second ionizing laser (termed MALDI-2). Proteins were digested in situ using trypsin prior to IMS analysis. For identification of peptides and proteins, a tissue homogenate was analyzed by LC-MS/MS for bottom-up proteomics and the corresponding proteins identified. These proteins were next fully ‘digested in silico’ to generate a database of theoretical peptides to then match to MALDI IMS datasets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list employing a 5 ppm error window. This resulted in 314 ± 45 (n=3) peptides and 1 112 ± 84 (n=3) peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to the LC-MS/MS peptide database. With positive protein identifications requiring two or more peptides per protein, 55 ± 13 proteins were identified with MALDI-1 and 205 ± 10 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.

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Enhancement of Tryptic Peptide Signals from Tissue Sections using MALDI IMS Post-ionization (MALDI-2)

10.26434/chemrxiv.13483302 ◽

2021 ◽

Author(s):

Josiah McMillen ◽

Danielle B. Gutierrez ◽

Audra M. Judd ◽

Jeffrey Spraggins ◽

Richard M. Caprioli

Keyword(s):

Imaging Mass Spectrometry ◽

Human Kidney ◽

Tissue Homogenate ◽

Laser Desorption Ionization ◽

Tissue Sections ◽

Enhanced Sensitivity ◽

Maldi Ims ◽

Peptide Database ◽

Peptide Signals

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization MALDI-2 has been shown to increase sensitivity for several molecular classes but thus far this has not been reported for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (termed MALDI-1), and conventional MALDI augmented using a second ionizing laser (termed MALDI-2). Proteins were digested in situ using trypsin prior to IMS analysis. For identification of peptides and proteins, a tissue homogenate was analyzed by LC-MS/MS for bottom-up proteomics and the corresponding proteins identified. These proteins were next fully ‘digested in silico’ to generate a database of theoretical peptides to then match to MALDI IMS datasets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list employing a 5 ppm error window. This resulted in 314 ± 45 (n=3) peptides and 1 112 ± 84 (n=3) peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to the LC-MS/MS peptide database. With positive protein identifications requiring two or more peptides per protein, 55 ± 13 proteins were identified with MALDI-1 and 205 ± 10 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.

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Enhancement of Tryptic Peptide Signals from Tissue Sections using MALDI IMS Post-ionization (MALDI-2)

10.26434/chemrxiv.13483302.v2 ◽

2021 ◽

Author(s):

Josiah McMillen ◽

Danielle B. Gutierrez ◽

Audra M. Judd ◽

Jeffrey Spraggins ◽

Richard M. Caprioli

Keyword(s):

Imaging Mass Spectrometry ◽

Human Kidney ◽

Tissue Homogenate ◽

Laser Desorption Ionization ◽

Tissue Sections ◽

Enhanced Sensitivity ◽

Maldi Ims ◽

Peptide Database ◽

Peptide Signals

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization MALDI-2 has been shown to increase sensitivity for several molecular classes but thus far this has not been reported for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (termed MALDI-1), and conventional MALDI augmented using a second ionizing laser (termed MALDI-2). Proteins were digested in situ using trypsin prior to IMS analysis. For identification of peptides and proteins, a tissue homogenate was analyzed by LC-MS/MS for bottom-up proteomics and the corresponding proteins identified. These proteins were next fully ‘digested in silico’ to generate a database of theoretical peptides to then match to MALDI IMS datasets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list employing a 5 ppm error window. This resulted in 314 ± 45 (n=3) peptides and 1 112 ± 84 (n=3) peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to the LC-MS/MS peptide database. With positive protein identifications requiring two or more peptides per protein, 55 ± 13 proteins were identified with MALDI-1 and 205 ± 10 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.

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MALDI TIMS IMS of Disialoganglioside Isomers – GD1a and GD1b in Murine Brain Tissue

10.33774/chemrxiv-2021-tkzc6 ◽

2021 ◽

Author(s):

Katerina Djambazova ◽

Martin Dufresne ◽

Lukasz Migas ◽

Angela Kruse ◽

Raf Van de Plas ◽

...

Keyword(s):

Mass Spectrometry ◽

Phase Separation ◽

Sialic Acid ◽

Gas Phase ◽

Sialic Acid Residue ◽

Spinal Cord Tissue ◽

Total Ganglioside ◽

Tissue Sections ◽

Maldi Ims ◽

Gas Phase Separation

Gangliosides are classified as acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or multiple sialic acid residue(s). The presence of multiple sialylation sites gives rise to highly diverse isomeric structures with distinct biological roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, directly from tissue sections. Integrating trapped ion mobility mass spectrometry (TIMS), a gas-phase separation technology, with MALDI IMS allows for the investi-gation of isomeric lipid structures in situ. Here we demonstrate the gas-phase separation of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in a standard mixture of both isomers, a total ganglioside extract, and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) were deter-mined from rat hippocampus, as well as in a spinal cord tissue section.

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Discovery of Prognostic Markers for Early-Stage High-Grade Serous Ovarian Cancer by Maldi-Imaging

Cancers ◽

10.3390/cancers12082000 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2000

Author(s):

Hagen Kulbe ◽

Oliver Klein ◽

Zhiyang Wu ◽

Eliane T. Taube ◽

Wanja Kassuhn ◽

...

Keyword(s):

Roc Analysis ◽

Operating Characteristic ◽

Prognostic Markers ◽

Early Stage ◽

Imaging Mass Spectrometry ◽

Maldi Imaging ◽

High Grade ◽

Proof Of Concept ◽

Tissue Sections ◽

Maldi Ims

With regard to relapse and survival, early-stage high-grade serous ovarian (HGSOC) patients comprise a heterogeneous group and there is no clear consensus on first-line treatment. Currently, no prognostic markers are available for risk assessment by standard targeted immunohistochemistry and novel approaches are urgently required. Here, we applied MALDI-imaging mass spectrometry (MALDI-IMS), a new method to identify distinct mass profiles including protein signatures on paraffin-embedded tissue sections. In search of prognostic biomarker candidates, we compared proteomic profiles of primary tumor sections from early-stage HGSOC patients with either recurrent (RD) or non-recurrent disease (N = 4; each group) as a proof of concept study. In total, MALDI-IMS analysis resulted in 7537 spectra from the malignant tumor areas. Using receiver operating characteristic (ROC) analysis, 151 peptides were able to discriminate between patients with RD and non-RD (AUC > 0.6 or < 0.4; p < 0.01), and 13 of them could be annotated to proteins. Strongest expression levels of specific peptides linked to Keratin type1 and Collagen alpha-2(I) were observed and associated with poor prognosis (AUC > 0.7). These results confirm that in using IMS, we could identify new candidates to predict clinical outcome and treatment extent for patients with early-stage HGSOC.

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Tryptic Peptide Signal Enhancement via MALDI IMS Post-ionization (MALDI-2) from Thin Tissue Sections

10.26434/chemrxiv.13483302.v1 ◽

2020 ◽

Author(s):

Josiah McMillen ◽

Danielle B. Gutierrez ◽

Audra M. Judd ◽

Jeffrey Spraggins ◽

Richard M. Caprioli

Keyword(s):

Imaging Mass Spectrometry ◽

Human Kidney ◽

Signal Enhancement ◽

Tissue Homogenate ◽

Data Sets ◽

Laser Desorption Ionization ◽

Tissue Sections ◽

Enhanced Sensitivity ◽

Maldi Ims

<a></a>Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the highly multiplexed, unlabeled mapping of analytes from thin tissue sections but further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization (MALDI-2) has been shown to increase sensitivity for numerous molecular classes for MALDI but this has not been demonstrated for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney with MALDI-2. Proteins were digested in situ using trypsin prior to the IMS analysis with MALDI (here, MALDI-1) and MALDI-2. For identification of peptides and proteins from MALDI IMS, a tissue homogenate was analyzed via LC-MS/MS for bottom-up proteomics and the proteins identified via LC-MS/MS were further ‘digested’ in silico to generate a database of theoretical peptides to match to MALDI IMS data sets. Peptides were tentatively identified by matching the MALDI peak list to the database within 5 ppm error that resulted in 170 ± 37 peptides and 885 ± 73 peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to LC-MS/MS results wherein positive identifications required two or more peptides to be detected per associated protein. This resulted in 55 ± 13 proteins identified with MALDI-1 and 205 ± 10 with MALDI-2. MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and it greatly increases the number of proteins identified during IMS experiments.

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Multiplex enzyme activity imaging by MALDI-IMS of substrate library conversions

Scientific Reports ◽

10.1038/s41598-020-72436-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Oliver Klein ◽

Akvile Haeckel ◽

Ulf Reimer ◽

Grit Nebrich ◽

Eyk Schellenberger

Keyword(s):

Enzyme Activities ◽

Imaging Mass Spectrometry ◽

Laser Desorption ◽

Biological Processes ◽

Laser Desorption Ionization ◽

Tissue Sections ◽

Enzyme Substrate ◽

Maldi Ims ◽

Enzyme Amplification ◽

Paraffin Tissue

Abstract Enzymes are fundamental to biological processes and involved in most pathologies. Here we demonstrate the concept of simultaneously mapping multiple enzyme activities (EA) by applying enzyme substrate libraries to tissue sections and analyzing their conversion by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). To that end, we spray-applied a solution of 20 naturally derived peptides that are known substrates for proteases, kinases, and phosphatases to zinc-fixed paraffin tissue sections of mouse kidneys. After enzyme conversion for 5 to 120 min at 37 °C and matrix application, the tissue sections were imaged by MALDI-IMS. We could image incubation time-dependently 16 of the applied substrates with differing signal intensities and 12 masses of expected products. Utilizing inherent enzyme amplification, EA-IMS can become a powerful tool to locally study multiple, potentially even lowly expressed, enzyme activities, networks, and their pharmaceutical modulation. Differences in the substrate detectability highlight the need for future optimizations.

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The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100118989 ◽

1985 ◽

Vol 43 ◽

pp. 426-429

Author(s):

George H. Herbener ◽

Antonio Nanci ◽

Moise Bendayan

Keyword(s):

Tissue Section ◽

Colloidal Gold ◽

Unit Area ◽

Protein A ◽

Extracellular Proteins ◽

Gold Complex ◽

Second Step ◽

Igg Antibodies ◽

Tissue Sections ◽

Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

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Discovery of intracellular 2,8 dihydroxyadenine crystals in bovine lymphatic and hepatic cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012881x ◽

1987 ◽

Vol 45 ◽

pp. 906-907

Author(s):

W. E. Rigsby ◽

D. M. Hinton ◽

V. J. Hurst ◽

P. C. McCaskey

Keyword(s):

Polarized Light ◽

Paraffin Sections ◽

Tissue Samples ◽

Whole Cells ◽

Tissue Sections ◽

Hepatic Cells ◽

Slaughter House ◽

Mammalian Tissues ◽

Carbon Coated ◽

Cellular Components

Crystalline intracellular inclusions are rarely seen in mammalian tissues and are often difficult to positively identify. Lymph node and liver tissue samples were obtained from two cows which had been rejected at the slaughter house due to the abnormal appearance of these organs in the animals. The samples were fixed in formaldehyde and some of the fixed material was embedded in paraffin. Examination of the paraffin sections with polarized light microscopy revealed the presence of numerous crystals in both hepatic and lymph tissue sections. Tissue sections were then deparaffinized in xylene, mounted, carbon coated, and examined in a Phillips 505T SEM equipped with a Tracor Northern X-ray Energy Dispersive Spectroscopy (EDS) system. Crystals were obscured by cellular components and membranes so that EDS spectra were only obtainable from whole cells. Tissue samples which had been fixed but not paraffin-embedded were dehydrated, embedded in Spurrs plastic, and sectioned.

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Ultrastructural aspects of olfactory signal transduction and its development

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016844x ◽

1994 ◽

Vol 52 ◽

pp. 142-143

Author(s):

Bert Ph. M. Menco

Keyword(s):

Signal Transduction ◽

Olfactory Receptor ◽

Receptor Cell ◽

Freeze Substitution ◽

Luminal Surface ◽

Tissue Sections ◽

Olfactory Receptor Cells ◽

Receptor Cells ◽

Olfactory Signal ◽

Odor Molecule

Vertebrate olfactory receptor cells are specialized neurons that have numerous long tapering cilia. The distal parts of these cilia line the interface between the external odorous environment and the luminal surface of the olfactory epithelium. The length and number of these cilia results in a large surface area that presumably increases the chance that an odor molecule will meet a receptor cell. Advanced methods of cryoprepration and immuno-gold labeling were particularly useful to preserve the delicate ultrastructural and immunocytochemical features of olfactory cilia required for localization of molecules involved in olfactory signal-transduction. We subjected olfactory tissues to freeze-substitution in acetone (unfixed tissues) or methanol (fixed tissues) followed by low temperature embedding in Lowicryl K11M for that purpose. Tissue sections were immunoreacted with several antibodies against proteins that are presumably important in olfactory signal-transduction.

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