Protein Identification Strategies in MALDI Imaging Mass Spectrometry
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Available online at www.sciencedirect.com ScienceDirect Protein identification strategies in MALDI imaging mass spectrometry: a brief review 1,2 1,2,3 Daniel J Ryan , Jeffrey M Spraggins and 1,2,3,4,5 Richard M Caprioli Matrix assisted laser desorption/ionization (MALDI) imaging specimens [1,2 ,3,4]. MALDI IMS allows for the label- mass spectrometry (IMS) is a powerful technology used to free, multiplex analysis of thousands of analytes across a investigate the spatial distributions of thousands of molecules samples surface yielding 2-dimensional molecular maps throughout a tissue section from a single experiment. As that elucidate both the localization and relative abundance proteins represent an important group of functional molecules of endogenous species. The technology has been used to in tissue and cells, the imaging of proteins has been an study awiderangeofanalyteclasses,includingmetabolites, important point of focus in the development of IMS drugs, lipids, peptides, and proteins [5,6 ,7 ,8]. The imag- technologies and methods. Protein identification is crucial for ing of proteins has garnered particular attention due to the the biological contextualization of molecular imaging data. role the proteins play in cellular processes [9], and because However, gas-phase fragmentation efficiency of MALDI MALDI IMS allows for the visualization of a protein and its generated proteins presents significant challenges, making various proteoforms (i.e. varying post-translational modifi- protein identification directly from tissue difficult. This review cations) in a single imaging experiment [10,11 ,12]. As highlights methods and technologies specifically related to highlighted in Figure 1, MALDI IMS is performed by first protein identification that have been developed to overcome coating a tissue section with a MALDI matrix, which assists these challenges in MALDI IMS experiments. in desorption and ionization of endogenous biomolecules during laser irradiation. Individual mass spectra are then collected at discrete x,y coordinates allowing for signal Addresses 1 intensity maps to be plotted across the sample area creating Department of Chemistry, Vanderbilt University, 7330 Stevenson ion images. A single MALDI IMS experiment can produce Center, Station B 351822, Nashville, TN 37235, USA 2 Mass Spectrometry Research Center, Vanderbilt University, 465 21st thousands of ion images, providing molecular context to Ave S #9160, Nashville, TN 37235, USA classical histological analysis. Fragmentation data is often 3 Department of Biochemistry, Vanderbilt University, 607 Light Hall, collected in separate experiments either directly from Nashville, TN 37205, USA 4 tissue [13] or by LC–MS/MS following extraction [14 ]. Department of Pharmacology, Vanderbilt University, 442 Robinson Research Building, 2220 Pierce Avenue, Nashville, TN 37232, USA 5 Department of Medicine, Vanderbilt University, 465 21st Ave S #9160, Protein identification in MALDI IMS is crucial in helping Nashville, TN 37235, USA to understand the physiological role of biomolecular and Corresponding author: cellular systems. However, identifying proteins observed Caprioli, Richard M ([email protected]) in many imaging experiments can be challenging because MALDI generated ions typically have low charge states (3), greatly reducing their gas-phase fragmentation effi- Current Opinion in Chemical Biology 2019, 48:64–72 ciency resulting in limited sequence coverage [15,16]. In This review comes from a themed issue on Omics general, protein identification in mass spectrometry is Edited by Ileana M Cristea and Kathryn S Lilley performed using either bottom-up or top-down sequenc- For a complete overview see the Issue and the Editorial ing [17]. Bottom-up protein identification methods rely on enzymatic digestion to hydrolyze larger proteins into Available online 23rd November 2018 smaller peptides that are easier to fragment, resulting in https://doi.org/10.1016/j.cbpa.2018.10.023 higher sequence coverage [18]. In top-down methods, 1367-5931/ã 2018 Elsevier Ltd. All rights reserved. intact proteins are injected into the mass spectrometer and subjected to fragmentation without prior digestion [19]. Aside from better tracking of protein modifications, an advantage of top-down protein sequencing is that it complements imaging experiments by enabling mass Introduction measurement of the intact protein that relates more directly to the MALDI IMS generated signals. Since the introduction of matrix-assisted laser desorption/ ionization imaging mass spectrometry (MALDI IMS) in the late 1990s, the technology has seen tremendous growth For both bottom-up and top-down proteomics experi- in utility, being employed to analyze biological substrates ments, proteins and peptides are commonly fragmented ranging from plants and insects, to mammalian tissues using collision induced dissociation (CID) or electron Current Opinion in Chemical Biology 2019, 48:64–72 www.sciencedirect.com Protein ID with MALDI IMS Ryan, Spraggins and Caprioli 65 Figure 1 (a) MALDI IMS Sample Preparation (b) MALDI IMS Acquisition x4, y1 a.u. 40 x3, y1 30 20 x2, y1 10 x1, y1 2000 3000 4000 5000 6000 7000 8000 9000 (c) Data Visualization 100% m/z 5654.36 m/z 5021.12 m/z 4936.52 0% (d) Analyte Identification Protein Abundance (%) Time (min) Current Opinion in Chemical Biology An overview of the MALDI IMS workflow for protein imaging. (a) A tissue sample is sectioned, washed to remove interfering salts and lipids, and coated homogenously with a MALDI matrix. After sample preparation, the sample is loaded into the instrument and the section is irradiated by a laser, moving a defined lateral distance which dictates the spatial resolution of the image. (b) A mass spectrum is generated at each pixel location. (c) Ion intensities for a selected mass range are then plotted in a coordinate system within the sampled tissue area, creating an ion image. (d) Following, or in parallel to IMS experiments, orthogonal experiments are completed in order to generate protein identifications that can be correlated to the imaging data. transfer dissociation (ETD). In CID, ions are accelerated fragments with poor sequence coverage. In ETD, ions and collide with a neutral gas leading to increased internal are bombarded with radical anions in an ion trap resulting energy of the ion. Should the deposited energy exceed the in electron transfer and formation of radical cations. Once critical energy of a bond, fragmentation will occur [20]. The the radical cation is formed, rapid dissociation along the ‘mobileproton model’ isused todescribethedissociationof peptide backbone occurs resulting in informative sequence proteins and peptides in CID studies [21]. This model fragments [22]. ETD requires that multiple protons are postulates that sequence fragments of highly charged pro- present, and the fragmentation efficiency for a given pro- teins result from charge directed fragmentation after the tein or peptide is highly correlated to an increased charge mobilization of a proton to a carbonyl on the peptide state. For these reasons, the applicability of ETD to backbone. However, MALDI primarily produces low MALDI generated proteins is also limited. charge state ions with few protons which tend to be seques- tered on highly basic amino acid side chains (e.g. Lys and To overcome the challenges associated with the identifi- Arg). Thus, MALDI generated protein ions produce cation of MALDI generated proteins, technologies and www.sciencedirect.com Current Opinion in Chemical Biology 2019, 48:64–72 66 Omics methods have been developed to enable separate, com- Fourier transform ion cyclotron IMS data set using mass plimentary proteomics experiments to be performed as accuracy [10]. Recently, a tissue homogenization protocol part of IMS workflows. These orthogonal experiments are was used in order to identify extracellular matrix proteins typically performed on the sample following MALDI using collagenase type III and elastase enzymes enabling image analysis or using a serial tissue section. In general, previously inaccessible collagens and elastin’s to be iden- these experiments involve the extraction of proteins from tified in IMS experiments [34 ]. Although tissue homog- the tissue with subsequent analysis of the sample by enization is the most utilized strategy for tissue proteo- electrospray ionization (ESI). ESI typically produces mics workflows, spatial fidelity is lost in most cases, highly charged ions that are more amenable to CID limiting its application for the analysis of discrete foci and ETD fragmentation [23]. By completing these in tissue. experiments offline, protein identifications can be made using more traditional proteomics workflows and the In-situ enzymatic digestions resulting identifications can be correlated to the imaging Ideally, proteins would be identified in an imaging exper- data through accurate mass matching [6 iment workflow from their host environment; that is, ,10,11 ,13,24,25]. directly from tissue. This allows both mass accuracy and spatial information to be used to relate imaging data Protein identification strategies in MALDI to protein identification experiments. As described above, imaging mass spectrometry workflows MALDI generated proteins suffer from inefficient frag- Analytical methods for generating protein identifications mentation in the gas phase. To overcome this challenge, in MALDI IMS workflows must balance trade-offs on-tissue enzymatic digestions can be performed to gen- between the effective spatial resolution