Examination of tumour tissues by direct MALDI-mass spectrometry imaging and profiling.

The purpose of the work described in this thesis was to develop and apply efficient methodologies based on MALDI-MSI for the direct analysis and targeting of protein tumour biomarkers within both frozen and formalin fixed paraffin embedded (FFPE) cancerous tissue sections.Method development for protein analysis directly in tumour tissue sections were performed using tumour xenograft models. This involved improvements in sample preparation, such as tissue washing protocols, and the development of data pre-processing methods prior to statistical analysis using a freely available software package, which referred to as Spec Align.The use of MALDI-MSI for studying proteome patterns directly from tumour tissue sections with no requirement for known targets is demonstrated. In addition, in situ identification of proteins within tumour tissue sections was achieved and correlated with their localisation. The method demonstrated here involved the use of octyl glucoside, a non-ionic detergent, which aims to improve the solubilisation and detection of low abundance and membrane-associated proteins within tumour tissue section after on-tissue digestion. The coupling of MALDI-MSI with ion mobility separation (IMS) has been found to improve the specificity and selectivity of the method.Combining these two methodological approaches allowed the targeting and identification of known tumour biomarkers and potential protein markers in various tumour tissue samples including frozen AQ4N dosed colon tumour xenografts and FFPE human adenocarcinoma tissue sections. The localisation and identification of proteins correlated to tumour growth and aggressiveness were studied using IMS-Tag MALDI-MSI, a novel concept developed in this work.In order to demonstrate its use as a potential biomarker discovery tool, MALDI-MSI was used for high throughput analysis of proteins within tissue micro arrays. Combining MALDI-MSI with statistical analysis allowed the design of a novel tumour classification model based on proteomic imaging information after on-tissue digestion.Another challenge for the MALDI-MSI technology is to achieve more targeted quantitative approaches for in situ analysis of proteins. A proof-of-concept based on multiple reaction monitoring (MRM) analysis with MALDI-MSI is described using a high repetition rate solid state laser. This aimed to improve the sensitivity and specificity of the methodology for the investigation of peptides/proteins directly within tumour tissue sections

Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed. 2014 American Chemical Societ

Examination of Tumour Tissues by Direct MALDI-Mass Spectrometry Imaging and Profiling

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Antigen retrieval prior to on-tissue digestion of formalin-fixed paraffin-embedded tumour tissue sections yields oxidation of proline residues.

MALDI-mass spectrometry imaging (MALDI-MSI) has been shown to allow the study of protein distribution and identification directly within formalin-fixed paraffin-embedded (FFPE) tissue sections. However, direct protein identification from tissue sections remains challenging due to signal interferences and/or existing post-translational or other chemical modifications. The use of antigen retrieval (AR) has been demonstrated for unlocking proteins prior to in situ enzymatic digestion and MALDI-MSI analysis of FFPE tissue sections. In the work reported here, the identification of proline oxidation, which may occur when performing the AR protocol, is described. This facilitated and considerably increased the number of identified peptides when adding proline oxidation as a variable modification to the MASCOT search criteria. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann

Targeting of Hypoxia in AQ4N-treated Tumour Xenografts by MALDI-Ion Mobility Separation-Mass Spectrometry Imaging.

In situ investigation and characterisation of hypoxia-related protein markers in AQ4N treated tumour xenografts. MALDI-IMS-MSI enabled the visualisation of the distribution of AQ4N and AQ4 within the tissue sections, hence the selective localisation hypoxic tissue regions. Protein distribution and identification were obtained directly from AQ4N treated and non treated tumour tissue sections after in situ digestion

Instrumentation and software for mass spectrometry imaging -making the most of what you've got.

Abstract not availablePaul J. Trim, Marie-Claude Djidja, Tasneem Muharib, Laura M. Cole, Bryn Flinders, Vikki A. Carolan, Simona Francese, Malcolm R. Clenc

Aggregation of Human Recombinant Monoclonal Antibodies Influences the Capacity of Dendritic Cells to Stimulate Adaptive T-cell Responses In Vitro

Subvisible proteinaceous particles which are present in all therapeutic protein formulations are in the focus of intense discussions between health authorities, academics and biopharmaceutical companies in the context of concerns that such particles could promote unwanted immunogenicity via anti-drug antibody formation. In order to provide further understanding of the subject, this study closely examines the specific biological effects proteinaceous particles may exert on dendritic cells (DCs) as the most efficient antigen-presenting cell population crucial for the initiation of the adaptive immune response. Two different model IgG antibodies were subjected to three different types of exaggerated physical stress to generate subvisible particles in far greater concentrations than the ones typical for the currently marketed biotherapeutical antibodies. The aggregated samples were used in in vitro biological assays in order to interrogate the early DC-driven events that initiate CD4 T-cell dependent humoral adaptive immune responses – peptide presentation capacity and co-stimulatory activity of DCs. Most importantly, antigen presentation was addressed with a unique approach called MHC-associated peptide proteomics (MAPPs), which allows for identifying the sequences of HLA-DR associated peptides directly from human dendritic cells [1]. The experiments demonstrated that highly aggregated solutions of two model mAbs generated under controlled conditions can induce activation of human monocyte-derived DCs as indicated by upregulation of typical maturation markers including co-stimulatory molecules necessary for CD4 T-cell activation. Additional data suggest that highly aggregated proteins could induce in vitro T-cell responses. Intriguingly, strong aggregation-mediated changes in the pattern and quantity of antigen-derived HLA-DR associated peptides presented on DCs were observed, indicating a change in protein processing and presentation. Increasing the amounts of subvisible proteinaceous particles correlated very well with the pronounced increase in the peptide number and clusters presented in the context of class II HLA-DR molecules, suggesting a major involvement of a mass-action mechanism of altering the presentation

Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed

MAPPs heat map of identified HLA-DR associated peptides in the HS study.

<p>Heat map visualization of mAb-derived HLA-DR associated peptides for both model antibodies in the HS study. Each sequence position is colored according to the presence and number of different mAb-derived peptides identified. In black colored sequence regions, no peptides were identified, in colored regions, peptides were identified, with the color coding for the number of different peptides identified per position. HS: aggregates generated by heat and shake stress; mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, un: unstressed, sl1: stress level 1, sl2: stress level 2.</p