Julien Franck

On-Tissue Protein Identification and Imaging by MALDI-Ion Mobility Mass Spectrometry

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues—formalin fixed paraffin embedded (FFPE) and frozen tissues—are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics “bottom-up” strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.

MALDI Imaging Mass Spectrometry STATE OF THE ART TECHNOLOGY IN CLINICAL PROTEOMICS

A decade after its inception, MALDI imaging mass spectrometry has become a unique technique in the proteomics arsenal for biomarker hunting in a variety of diseases. At this stage of development, it is important to ask whether we can consider this technique to be sufficiently developed for routine use in a clinical setting or an indispensable technology used in translational research. In this report, we consider the contributions of MALDI imaging mass spectrometry and profiling technologies to clinical studies. In addition, we outline new directions that are required to align these technologies with the objectives of clinical proteomics, including: 1) diagnosis based on profile signatures that complement histopathology, 2) early detection of disease, 3) selection of therapeutic combinations based on the individual patients entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.

Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin-fixed paraffin-embedded adenocarcinoma tissue sections

The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI‐mass spectrometry imaging (MALDI‐MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin‐fixed paraffin‐embedded breast cancer tissue sections were used. An improved protocol for on‐tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin‐fixed paraffin‐embedded tumour tissue sections. A novel approach combining MALDI‐MSI and ion mobility separation MALDI‐tandem mass spectrometry imaging for improving the detection of low‐abundance proteins that are difficult to detect by direct MALDI‐MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI‐MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified.

Liquid ionic matrixes for MALDI mass spectrometry imaging of lipids

Lipids are a major component of cells and play a variety of roles in organisms. In general, they play a key role in the structural composition of membranes. Some lipids, such as sphingoglycolipids, however, are also mediators of different biological processes, including protein transport, regulation of cell growth, cellular morphogenesis, neuronal plasticity, and regulation of the immune response. With the advent of MALDI mass spectrometry imaging (MALDI MSI), lipids have begun to be intensively investigated by several groups. Here we present a novel development in the detection and study of lipids using an automatic microspotter coupled to specific liquid ionic matrixes based on a 2,5-DHB matrix (i.e., 2,5-DHB/ANI, 2,5-DHB/Pyr, and 2,5-DHB/3-AP). This development allows to decrease the time of the sample preparation by comparison with crystalline 2,5-DHB as matrix and was validated on human ovarian cancer biopsies to demonstrate its use as a precise procedure that is particularly useful for specific diagnoses.

MALDI imaging and profiling MS of higher mass proteins from tissue

MALDI imaging and profiling mass spectrometry of proteins typically leads to the detection of a large number of peptides and small proteins but is much less successful for larger proteins: most ion signals correspond to proteins of m/z < 25,000. This is a severe limitation as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights exceeding 25 kDa. The detector technology typically used for protein imaging, a microchannel plate, is not well suited to the detection of high m/z ions and is prone to detector saturation when analyzing complex mixtures. Here we report increased sensitivity for higher mass proteins by using the CovalX high mass HM1 detector (Zurich, Switzerland), which has been specifically designed for the detection of high mass ions and which is much less prone to detector saturation. The results demonstrate that a range of different sample preparation strategies enable higher mass proteins to be analyzed if the detector technology maintains high detection efficiency throughout the mass range. The detector enables proteins up to 70 kDa to be imaged, and proteins up to 110 kDa to be detected, directly from tissue, and indicates new directions by which the mass range amenable to MALDI imaging MS and MALDI profiling MS may be extended.

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer

The large amount of data generated using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) poses a challenge for data analysis. In fact, generally about 1.108–1.109 values (m/z, I) are stored after a single MALDI-MSI experiment. This imposes processing techniques using dedicated informatics tools to be used since manual data interpretation is excluded. This work proposes and summarizes an approach that utilizes a multivariable analysis of MSI data. The multivariate analysis, such as principal component analysis–symbolic discriminant analysis, can remove and highlight specific m/z from the spectra in a specific region of interest. This approach facilitates data processing and provides better reproducibility, and thus, broadband acquisition for MALDI-MSI should be considered an effective tool to highlight biomarkers of interest. Additionally, we demonstrate the importance of the hierarchical classification of biomarkers by analyzing studies of clusters obtained either from digested or undigested tissues and using bottom-up and in-source decay strategies for in-tissue protein identification. This provides the possibility for the rapid identification of specific markers from different histological samples and their direct localization in tissues. We present an example from a prostate cancer study using formalin-fixed paraffin-embedded tissue.

Improving tissue preparation for matrix-assisted laser desorption ionization mass spectrometry imaging. Part 1: using microspotting.

Nowadays, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is a powerful technique to obtain the distribution of endogenous and exogenous molecules within tissue sections. It can, thus, be used to study the evolution of molecules across different physiological stages in order to find out markers or get knowledge on signaling pathways. In order to provide valuable information, we must carefully control the sample preparation to avoid any delocalization of molecules of interest inside the tissue during this step. Currently, two strategies can be used to deposit chemicals, such as the MALDI matrix, onto the tissue both involving generation of microdroplets that will be dropped off onto the surface. First strategy involves microspraying of solutions. Here, we have been interested in the development of a microspotting strategy, where nanodroplets of solvent are ejected by a piezoelectric device to generate microspots at the tissue level. Such systems allow one to precisely control sample preparation by creating an array of spots. In terms of matrix crystallization, a microspotting MALDI matrix is hardly compatible with the results by classical (pipetting) methods. We have thus synthesized and studied new solid ionic matrixes in order to obtain high analytical performance using such a deposition system. These developments have enabled optimization of the preparation time because of the high stability of the printing that is generated in these conditions. We have also studied microspotting for performing on-tissue digestion in order to go for identification of proteins or to work from formalin fixed and paraffin embedded (FFPE) tissue samples. We have shown that microspotting is an interesting approach for on tissue digestion. Peptides, proteins, and lipids were studied under this specific preparation strategy to improve imaging performances for this class of molecules.

Development of liquid microjunction extraction strategy for improving protein identification from tissue sections

MALDI Mass Spectrometry Imaging has shown important potential for molecular classification and pathology marker discovery. Protein markers identification is therefore of prime importance. Direct structural analysis from tissue sections has shown limitations for protein identification because of the high degree of complexity of tissues. Only proteins of major abundance are identified this way. On the contrary, conventional proteomics approaches clearly allow for reliable identification of complex protein extracts but do not provide fine correlation with protein location in their original context. Here is presented an approach to obtain identification of proteins of various abundances while keeping their localization within the section. On-tissue trypsin digestion followed by micro-extraction using a liquid micro-junction interface is an efficient strategy to extract tryptic peptides and further identify the associated proteins off tissues. It was shown that conventional Reverse Phase Liquid Chromatography separation on the extracted material followed by MS/MS analysis on a HR FTMS instrument enabled the identification of 1500 proteins on average with high confidence from an area of about 650μm in diameter, which corresponds to an estimated number of 1900 cells in average. The approach can be easily integrated in the MALDI MSI workflow and should provide interesting insights for clinical applications.

On-Tissue N-Terminal Peptide Derivatizations for Enhancing Protein Identification in MALDI Mass Spectrometric Imaging Strategies

Matrix-assisted laser desorption/ionization (MALDI) is a new tool that can acquire the localization of various compounds, including peptides and proteins, directly from tissue sections. Despite the important developments recently performed in the field of MALDI imaging in tissue, the precise identification of compounds still needs improvement. We have developed N-terminal chemical derivatization strategies to improve tissue identification of proteins, including de novo sequencing performance. We have first focused on sulfonation agents, such as 4-SPITC and 3-SBASE. These two derivatizations were optimized to be performed directly on tissue sections. By adding a negative charge at the N-terminus of a tryptic digest peptide, we were able to generate a complete y fragment series directly from the tissue. Of these derivatizations, 3-SBASE has shown to be more efficient, as loss of the derivative group is one of the major fragmentation pathways for 4-SPITC. 3-SBASE was optimized so that the derivatization reaction could be automatically performed using an automatic microspotting device. It was then included in an automatic process that included automated trypsin digestion and matrix deposition. Derivatizations allowed the acquisition to be easily interpretable by MS(2) spectra, leading to very precise identification as well as easy manual reading of sequences for de novo sequencing. It was observed that only arginine-terminated peptides were observed after derivatization, likely due to the high gas-phase basicity of such peptides compared to those that are lysine-terminated. We also observed a stop in the y fragmentation series for peptides presenting a miscleavage. We have now begun to study a different derivatization using N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This derivatization allows the orientating of a fragmentation toward a series of fragment ions, and thus it is independent of the presence of basic residues in the sequence. This derivatization can be performed at room temperature, which greatly facilitates the automation of the process. The TMPP derivatization therefore yields an advantageous new generation of derivatives suited for use in tissue.

MALDI Direct Analysis and Imaging of Frozen Versus FFPE Tissues: What Strategy for Which Sample?

Significant advances have been made in the past decade in the field of mass spectrometry imaging with MALDI ion sources (MALDI-MSI). While MALDI-MSI has high potential in the field of biology and in the clinic, a challenge for MALDI-MSI has been to adapt itself to a greater range of sample types. In particular, much of the biological archived materials for pathology studies are tissue biopsies fixed with paraformaldehyde and embedded in paraffin (FFPE tissues) because of the high stability of such samples. Thus, there has been a need to develop strategies for analyzing FFPE samples as this would allow retrospective studies of past clinical cases on large cohorts of existing samples. Obviously, PAF fixation, by inducing protein cross-linking, causes problems for molecular analysis by MS. We developed on tissue digestion strategies for overcoming these difficulties and allowing molecular data to be retrieved from FFPE samples no matter how long they have been stored. These digestion strategies preserve localization from digested proteins making MALDI-MSI of proteins possible by monitoring the resulting peptides. We present methods and protocols for FFPE samples. These strategies have proven to be valuable for all tested FFPE samples and have opened archived tissues from hospital banks to MALDI-MSI.