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

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 <i>in situ</i> 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 <i>in silico’</i> 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.<br>

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

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 <i>in situ</i> 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 <i>in silico’</i> 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.<br>

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

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 <i>in situ</i> 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 <i>in silico’</i> 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.<br>

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

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

<p><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 <i>in situ</i> 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’ <i>in silico</i> 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.<b></b></p>

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

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.

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

Cancers ◽  
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.

scholarly journals Resolving the Complexity of Spatial Lipidomics Using MALDI TIMS Imaging Mass Spectrometry

2020 ◽  
Author(s):  
Katerina Djambazova ◽  
Dustin R. Klein ◽  
Lukasz Migas ◽  
Elizabeth Neumann ◽  
Emilio Rivera ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Acyl Chain ◽  
Imaging Mass Spectrometry ◽  
Structural Diversity ◽  
Data Interpretation ◽  
Whole Body ◽  
Tissue Sections ◽  
Mouse Pup

<p>Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct map-ping of biomolecules in tissue. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only <i>m/z </i>information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables approximately a ~270% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn-backbone isomers, acyl chain isomers, as well as double bond positional and geometric isomers are demonstrated. As a proof-of-concept, <i>in situ </i>separation and imaging of lipid isomers with distinct spatial distributions was performed using tissue sections from a whole-body mouse pup.</p>

scholarly journals Lipidoak aztertu eta identifikatzeko TLC kromatografiaren eta MALDI masa-espektrometriaren arteko akoplamendua

2018 ◽  
pp. 149-165
Author(s):  
Antonio Veloso
Keyword(s):  
Mass Spectrometry ◽  
Imaging Mass Spectrometry ◽  
Laser Desorption ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Maldi Ims ◽  
Layer Chromatography ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

Geruza meheko kromatografia teknika (TLC, ingelesezko Thin Layer Chromatography) oso erabilia izan da hainbat molekularen banaketa eta karakterizaziorako. Bestalde, Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) edo matrizez lagundutako laserraren bidezko desortzioionizazioa/hegaldi-denbora masa-espektrometria teknikaren erabilera gero eta gehiago hedatzen ari da molekula desberdinen analisia egiteko. Gainera, Imaging Mass Spectrometry (IMS) edo masa-espektrometriaren bidezko irudia izeneko teknikak, ahalbidetzen du ehun biologikoetan dauden lipidoen, proteinen eta farmakoen banaketa-irudiak lortzea eta teknika bikaina da molekula horien banaketa aztertzeko. Teknika honek lipido mota ezberdinen identifikazioa eta lokalizazioa ahalbidetzen du, konposatuaren edota familia kimikoaren aukera egin aurretik. Analizatu behar diren konposatuen aukera aldez aurretik egin behar ez denez, aukera dago irudiak sortzeko masa-espektrometroan detektatutako ioietatik abiatuta. Lan honetan, erakusten da lipidoen ikerkuntzarako oso lagungarria dela TLC xaflak eta MALDI IMS teknikak konbinatzea.

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

2021 ◽  
Author(s):  
Josiah C. McMillen ◽  
Danielle B. Gutierrez ◽  
Audra M. Judd ◽  
Jeffrey M. Spraggins ◽  
Richard M. Caprioli
Keyword(s):  
Tryptic Peptide ◽  
Tissue Sections ◽  
Maldi Ims ◽  
Peptide Signals
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