Laser capture microdissection coupled mass spectrometry (LCM-MS) for spatially resolved analysis of formalin-fixed and stained human lung tissues

From Springer Nature via Jisc Publications RouterHistory: received 2019-11-06, accepted 2020-06-11, registration 2020-06-11, pub-electronic 2020-06-17, online 2020-06-17, pub-print 2020-12Publication status: PublishedFunder: Wellcome Trust; doi: http://dx.doi.org/10.13039/100004440; Grant(s): 203128/Z/16/ZFunder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/L024551/1Abstract: Background: Haematoxylin and eosin (H&E)—which respectively stain nuclei blue and other cellular and stromal material pink—are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing and H&E staining of tissues provides challenges with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique to analyse H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. Methods: Herein, we utilize heat extraction, physical disruption, and in column digestion for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE-fixed H&E-stained sections from Idiopathic Pulmonary Fibrosis (IPF) specimens (n = 3 IPF specimens) were then subject to a qualitative and then quantitative proteomics approach using BayesENproteomics. In addition, we tested the sensitivity of this method by processing and analysing a range of micro-dissected human lung blood vessel tissue volumes. Results: This approach yields 1252 uniquely expressed proteins (at a protein identification threshold of 3 unique peptides) with 892 differentially expressed proteins between these regions. In accord with prior knowledge, our methodology approach confirms that human lung blood vessels are enriched with smoothelin, CNN1, ITGA7, MYH11, TAGLN, and PTGIS; whereas morphologically normal human lung alveoli are enriched with cytokeratin-7, -8, -18, -19, 14, and -17. In addition, we identify a total of 137 extracellular matrix (ECM) proteins and immunohistologically validate that laminin subunit beta-1 localizes to morphologically normal human lung alveoli and tenascin localizes to human lung blood vessels. Lastly, we show that this micro-proteomics technique can be applied to tissue volumes as low as 0.0125 mm3. Conclusion: Herein we show that our multistep sample preparation methodology of LCM-MS can identify distinct, characteristic proteomic compositions of anatomical features within complex fixed and stained tissues

Peptide location fingerprinting reveals modification‐associated biomarker candidates of ageing in human tissue proteomes

From Wiley via Jisc Publications RouterHistory: received 2020-10-08, rev-recd 2021-02-18, accepted 2021-03-15, pub-electronic 2021-04-08, pub-print 2021-05Article version: VoRPublication status: PublishedFunder: Walgreens Boots AllianceAbstract: Although dysfunctional protein homeostasis (proteostasis) is a key factor in many age‐related diseases, the untargeted identification of structurally modified proteins remains challenging. Peptide location fingerprinting is a proteomic analysis technique capable of identifying structural modification‐associated differences in mass spectrometry (MS) data sets of complex biological samples. A new webtool (Manchester Peptide Location Fingerprinter), applied to photoaged and intrinsically aged skin proteomes, can relatively quantify peptides and map statistically significant differences to regions within protein structures. New photoageing biomarker candidates were identified in multiple pathways including extracellular matrix organisation (collagens and proteoglycans), protein synthesis and folding (ribosomal proteins and TRiC complex subunits), cornification (keratins) and hemidesmosome assembly (plectin and integrin α6β4). Crucially, peptide location fingerprinting uniquely identified 120 protein biomarker candidates in the dermis and 71 in the epidermis which were modified as a consequence of photoageing but did not differ significantly in relative abundance (measured by MS1 ion intensity). By applying peptide location fingerprinting to published MS data sets, (identifying biomarker candidates including collagen V and versican in ageing tendon) we demonstrate the potential of the MPLF webtool for biomarker discovery

Syndecan-4 phosphorylation is a control point for integrin recycling

Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration

Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly

Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is currently lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this dataset reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome likely represents a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community

The effect of peptide adsorption on signal linearity and a simple approach to improve reliability of quantification

Peptide quantification using MS often relies on the comparison of peptide signal intensities between different samples, which is based on the assumption that observed signal intensity has a linear relationship to peptide abundance. A typical proteomics experiment is subject to multiple sources of variance, so we focussed here on properties affecting peptide linearity under simple, well-defined conditions. Peptides from a standard protein digest were analysed by multiple reaction monitoring (MRM) MS to determine peptide linearity over a range of concentrations. We show that many peptides do not display a linear relationship between signal intensity and amount under standard conditions. Increasing the organic content of the sample solvent increased peptide linearity by increasing the accuracy and precision of quantification, which suggests that peptide non-linearity is due to concentration-dependent surface adsorption. Using multiple peptides at various dilutions, we show that peptide non-linearity is related to observed retention time and predicted hydrophobicity. Whereas the effect of adsorption on peptide storage has been investigated previously, here we demonstrate the deleterious effect of peptide adsorption on the quantification of fresh samples, highlight aspects of sample preparation that can minimise the effect, and suggest bioinformatic approaches to enhance the selection of peptides for quantification. BIOLOGICAL SIGNIFICANCE: Accurate quantification is central to many aspects of science, especially those examining dynamic processes or comparing molecular stoichiometries. In biological research, the quantification of proteins is an important yet challenging objective. Large-scale quantification of proteins using MS often depends on the comparison of peptide intensities with only a single-level calibrant (as in stable isotope labelling and absolute quantification approaches) or no calibrants at all (as in label-free approaches). For these approaches to be reliable, it is essential that the relationship between signal intensity and concentration is linear, without a significant intercept. Here, we show that peptide adsorption can severely affect this relationship, even under controlled conditions, and we demonstrate simple methodologies that can be used to moderate and predict this effect. These findings thus enable the quantification of proteins with increased robustness and reliability

Dynamic modification of the ETS transcription factor PEA3 by sumoylation and p300-mediated acetylation.

Transcription factor activity is often controlled through the dynamic use of post-translational modifications. Two such modifications are acetylation and sumoylation, which both occur on lysine residues, providing the opportunity for cross-talk at the molecular level. Here, we focussed on the ETS-domain transcription factor PEA3 and studied the potential interplay between these two modifications. We demonstrate that PEA3 is acetylated in a p300-dependent manner. ERK MAPK pathway signalling potentiates acetylation of PEA3, and enhances its trans-activation capacity. However, the major acetylation and sumoylation events take place on the same sites in PEA3 making simultaneous modification impossible. Indeed, manipulation of either the sumoylation or acetylation pathways causes reciprocal changes in PEA3 acetylation and sumoylation respectively. However, despite the mutually exclusive nature of these modifications, both contribute to the trans-activation capacity of PEA3, implying that a dynamic series of modification events occurs during the activation process

Tyrannosaurus rex Osborn 1905

(a) <i>Tyrannosaurus rex</i> collagen sequences <p> In the case of the <i>T. rex</i> specimen (MOR 1125)̗ these initial studies were supported by two main lines of supporting molecular evidence: immunological data and sequence information [1]. This first choice of support is no longer widely accepted as ideal for such claims̗ given that immunological techniques have been shown to yield false-positive results [8]. The authenticity of any findings based on this approach rests solely on sequence interpretation. In its first release̗ this was fraught with multiple incorrect post-translational modification (PTM) assignments in the form of hydroxylated glycine residues [5]; a clear indication of the potential problems is the reliance on probability-based matching algorithms of current proteomics-based techniques. In direct response to this first report̗ several criticisms arose related to potential forms of contamination [6 ̗ 9 ̗ 10] or statistical artefact relating to such a proteomics approach [11].</p> <p> On the grounds that mineralized and non-mineralized coatings have been found extensively in the porous trabecular bone of a variety of vertebrate fossils across time̗ including dinosaurs̗ Kaye <i>et al.</i> [10] proposed that the <i>T. rex</i> specimen was likely similarly contaminated with bacterial biofilm̗ thus explaining the morphological similarity to the blood vessels and osteocytes that they attack. In addition̗ the blood-celllike iron–oxygen spheres found in the vessels were identified as an oxidized form of formerly pyritic framboids. Interestingly̗ similar thin linings on Haversian canals within apatite were identified by infrared and electron microprobe analysis of ossified tendon by Manning <i>et al.</i> [7] and clearly showed preserved mineral zonation̗ with silica and trapped carbon dioxide. FTIR analysis of the tendon showed clear structural control of organic compounds within the Haversian canals̗ suggesting that organic material may have persisted. However̗ this study concluded that the organic signal may have been associated with breakdown products of the original biomaterial deposited within the tendon̗ consistent with the presence of the endogenous breakdown products of organic material identified from other regions of the specimen but not able to yield any such sequence information [7].</p> <p> Bern <i>et al.</i> [9] reanalysed the original <i>T. rex</i> sequence data to infer that the sample was predominantly laboratory contaminants̗ soil bacteria and bird-like haemoglobin and collagen. They suggested that of the six peptides that Asara <i>et al.</i> [5] deposited in GenBank (GATGAPGIAGAPGFPGARGA- PGPQGPSGAPGPK̗ GSAGPPGATGFPGAAGR̗ GVQG- PPGPQGPR̗ and GVVGLPGQR from collagen alpha-I type I̗ GLVGAPGLRGLPGK from collagen alpha-1 type II and GLPGESGAVGPAGPIGSR from collagen alpha-2 type I)̗ only the first three of these could be considered statistically significant̗ calling for the latter to be dropped from GenBank. However̗ despite the unexpected presence of haemoglobin̗ a protein only typically seen in relatively recent samples [12̗ 13]̗ the presence of the remaining collagen peptides was not accepted as being contamination for reasons that remain unclear.</p>Published as part of <i>Michael Buckley, Stacey Warwood, Bart van Dongen, Andrew C. Kitchener & Phillip L. Manning, 2017, A fossil protein chimera; difficulties in discriminating dinosaur peptide sequences from modern cross-contamination, pp. 544 in Proceedings of the Royal Society, Series B 284</i> on page 544, DOI: 10.1098/rspb.2017.0544, <a href="http://zenodo.org/record/3748698">http://zenodo.org/record/3748698</a&gt

A fossil protein chimera; difficulties in discriminating dinosaur peptide sequences from modern cross-contamination

A decade ago, reports that organic-rich soft tissue survived from dinosaur fossils were apparently supported by proteomics-derived sequence information of exceptionally well-preserved bone. This initial claim to the sequencing of endogenous collagen peptides from an approximately 68 Myr Tyrannosaurus rex fossil was highly controversial, largely on the grounds of potential contamination from either bacterial biofilms or from laboratory practice. In a subsequent study, collagen peptide sequences from an approximately 78 Myr Brachylophosaurus canadensis fossil were reported that have remained largely unchallenged. However, the endogeneity of these sequences relies heavily on a single peptide sequence, apparently unique to both dinosaurs. Given the potential for cross-contamination from modern bone analysed by the same team, here we extract collagen from bone samples of three individuals of ostrich, Struthio can-telus. The resulting LC MS/MS data were found to match all of the proposed sequences for both the original Tyrannosaurus and Brachylophosaurus studies. Regardless of the true nature of the dinosaur peptides, our finding highlights the difficulty of differentiating such sequences with confidence. Our results not only imply that cross-contamination cannot be ruled out, but that appropriate measures to test for endogeneity should be further evaluated

Brachylophosaurus canadensis Sternberg 1953

(b) <i>Brachylophosaurus canadensis</i> collagen sequences <p> Following the initial 2007 report [5]̗ the same team reported similar collagen peptide sequence matches from a hadrosaurine dinosaur̗ an approximately 78 Ma <i>Brachylophosaurus canadensis</i> (MOR 2598; table 1) [14]. However̗ although it had already been suggested that standards be set in place̗ like those for the field of ancient DNA̗ this second study once again aimed to rely on an immunological approach as the main line of support̗ despite the ability to record chemical decay within proteins through PTMs such as oxidations and deamidations [15 – 17]̗ or even a range of others identified by the same team in ancient moa [18].</p> <p>*Note that these were the two peptides observed in both analyses [5].</p> <p> Following Schweitzer <i>et al.</i> [14]̗ there have been no further published attempts to verify the endogeneity of either published samples of purported dinosaur collagen sequences from other research groups̗ despite the lack of potential means to clarify the extent of decay within the proteins̗ of which we would expect substantial alteration [7]; members of the same team subsequently went on to report even more exceptional peptide matches to soft-tissue structures̗ in which they interestingly did report on the levels of deamidation and made clear attempts to separate modern from fossil material during the laboratory process [19]. The published record to date could be considered to lean in favour of endogeneity̗ with Peterson <i>et al.</i> [20] arguing against the microbial biofilm interpretation̗ suggesting that the crystallization of microbial biofilms on decomposing organic matter within vertebrate bone in early taphonomic stages may contribute to the preservation of primary soft tissues deeper in the bone structure [14].</p> <p> A subsequent study mapping the molecular locations of the matched collagen peptides from both dinosaurs also implied that it was functionally significant regions of the collagen fibrils that were matched [21]. Although it was suggested that this non-random distribution could support the hypothesis that the peptides are produced from the extinct organisms̗ while also suggesting a chemical mechanism for survival̗ it does not rule out cross-contamination in which the same ‘mechanism for survival’ could equally apply to enhanced likelihood of contaminant peptides. More recently̗ a second collagen-based study has been published that placed further emphasis on the cleaning of the instrumentation used in addition to separate laboratories for extant and fossil material [22]̗ presenting an overlapping set of peptides. Intriguingly̗ these do not include the peptide sequence found as unique to both dinosaurs (table 2). As a result̗ the phylogenetic analysis of this latest extraction places the <i>Brachylophosaurus</i> as sister-group to alligators as well [22]̗ clearly highlighting concern regarding the limitations of the study to date. They do̗ however̗ all match with peptides from alligator type 1 collagen̗ a species concurrently analysed in their previous works as modern reference material [5 ̗ 14] even if not necessarily contamination caused at the time of the most recent sampling.</p> <p> Given that the only reports that appear to favour the most recent studies cannot rule out cross-contamination̗ we set out to test whether or not the reported set of unique collagen peptides (i.e. [5 ̗ 14]̗ excluding [22] as not containing unique peptides) could simply reflect cross-sample contamination from the modern reference material used; in this case̗ ostrich (<i>S. camelus</i>) bone (alligator was also used in the latter study̗ but not evaluated here in determining the unique dinosaur peptide because it was not used in the earlier study). In this study̗ we aimed to investigate the differences between sequences from ostrich bone collagen and those reported for both <i>T. rex</i> (MOR 1125) and <i>B. canadensis</i> (MOR 2598).</p>Published as part of <i>Michael Buckley, Stacey Warwood, Bart van Dongen, Andrew C. Kitchener & Phillip L. Manning, 2017, A fossil protein chimera; difficulties in discriminating dinosaur peptide sequences from modern cross-contamination, pp. 544 in Proceedings of the Royal Society, Series B 284</i> on page 544, DOI: 10.1098/rspb.2017.0544, <a href="http://zenodo.org/record/3748698">http://zenodo.org/record/3748698</a&gt