Use of tryptic peptide MALDI mass spectrometry imaging to identify the spatial proteomic landscape of colorectal cancer liver metastases.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. CRC liver metastases (CRLM) are often resistant to conventional treatments, with high rates of recurrence. Therefore, it is crucial to identify biomarkers for CRLM patients that predict cancer progression. This study utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially map the CRLM tumour proteome. CRLM tissue microarrays (TMAs) of 84 patients were analysed using tryptic peptide MALDI-MSI to spatially monitor peptide abundances across CRLM tissues. Abundance of peptides was compared between tumour vs stroma, male vs female and across three groups of patients based on overall survival (0-3 years, 4-6 years, and 7+ years). Peptides were then characterised and matched using LC-MS/MS. A total of 471 potential peptides were identified by MALDI-MSI. Our results show that two unidentified m/z values (1589.876 and 1092.727) had significantly higher intensities in tumours compared to stroma. Ten m/z values were identified to have correlation with biological sex. Survival analysis identified three peptides (Histone H4, Haemoglobin subunit alpha, and Inosine-5'-monophosphate dehydrogenase 2) and two unidentified m/z values (1305.840 and 1661.060) that were significantly higher in patients with shorter survival (0-3 years relative to 4-6 years and 7+ years). This is the first study using MALDI-MSI, combined with LC-MS/MS, on a large cohort of CRLM patients to identify the spatial proteome in this malignancy. Further, we identify several protein candidates that may be suitable for drug targeting or for future prognostic biomarker development.

High-Resolution N-Glycan MALDI Mass Spectrometry Imaging of Subchondral Bone Tissue Microarrays in Patients with Knee Osteoarthritis.

N-glycan alterations contribute to the progression of several joint diseases, including knee osteoarthritis (KOA). However, molecular changes in KOA subchondral trabecular bone, when exposed to different joint loading forces, are still unknown. The aim of this study was, therefore, to demonstrate the feasibility to differentiate N-glycan changes in subchondral trabecular bone from four different joint loading forces of the tibial plateau regions (i.e., Lateral Anterior (L-A), Lateral Posterior (L-P), Medial Anterior (M-A), and Medial Posterior (M-P)) in KOA patients (n = 10) using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at 20 µm spatial resolution. The degree of cartilage degeneration was evaluated histologically, and the subchondral bone tissue microarrays (TMAs) were subsequently manually constructed from formalin-fixed paraffin-embedded (FFPE) KOA osteochondral (i.e., cartilage-subchondral bone) tissues. Overall, the Osteoarthritis Research Society International (OARSI) histological grade was significantly higher and the size of chondrocytes in the superficial zone was much larger for both M-A and M-P compared to L-A and L-P of cartilage (p = 0.006, p = 0.030, p = 0.028, and p = 0.010; respectively). Among the 65 putative N-glycans observed by MALDI-MSI, 2 core fucosylated bi-antennary N-glycans, m/z 1809.64; (Hex)5(HexNAc)4(Fuc)1 and 2100.73; (NeuAc)1(Hex)5(HexNAc)4(Fuc)1, were significantly higher in intensity in M-A compared to L-A of the trabecular bone (p = 0.027, and p = 0.038, respectively). These N-glycans were then further structurally characterized by in situ MS/MS fragmentation post-MALDI-MSI. Our results demonstrate, for the first time, N-glycan alterations can occur at different joint loading forces in the KOA tibial plateau and the feasibility of subchondral bone TMA construction for N-glycan MALDI-MSI.

Complex-Type N-Glycans Are Associated with Cartilage Degeneration within Different Loading Sites of the Tibial Plateau for Knee Osteoarthritis Patients.

Abnormal N-glycosylation has been shown to play an important role in the pathogenesis of multiple diseases. However, little is known about the relationship between N-glycosylation and knee osteoarthritis (KOA) progression at the tissue level. Thus, the aim of this study was to quantify the cartilage histomorphometric changes in formalin-fixed paraffin-embedded (FFPE) tissue collected from the lateral and medial compartments of the tibial plateau KOA patients (n = 8). Subsequently, N-glycans were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) followed by in situ MS/MS fragmentation. Overall, the Osteoarthritis Research Society International (OARSI) histological grade and cartilage surface fibrillation index were significantly higher, and chondrocyte size in the superficial zone was much larger, for the medial high-loaded cartilage compared to the lateral less-loaded cartilage. Among 92 putative N-glycans observed by MALDI-MSI, 3 complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, and 1 oligomannose-type N-glycan, (Hex)9(HexNAc)2, were significantly higher in intensity in the medial cartilage compared to the lateral cartilage, whereas 2 tetra-antennary fucosylated-type N-glycans, (Hex)3(HexNAc)6(Fuc)2 and (Hex)3(HexNAc)6(Fuc)3, were significantly higher in intensity in the lateral cartilage than the medial cartilage. Our findings indicate that complex-type N-glycans are associated with higher severity of cartilage degeneration and may influence the cellular processes of KOA.

Mass spectrometry imaging spatially identifies complex-type N-glycans as putative cartilage degradation markers in human knee osteoarthritis tissue.

N-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N-glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (n=3) and CTL individuals (n=3), aged >55 years old, were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N-glycans could further be evaluated as potential diagnostic and prognostic biomarkers.

Greater heterogeneity of the bone mineralisation density distribution and low bone matrix mineralisation characterise tibial subchondral bone marrow lesions in knee osteoarthritis patients.

Tibial subchondral bone marrow lesions (BMLs) identified by MRI have been recognised as potential disease predictors in knee osteoarthritis (KOA), and may associate with abnormal bone matrix mineralisation and reduced bone quality. However, these tissue-level changes of BMLs have not been extensively investigated. Thus, the aim of this study was to quantify the degree of subchondral bone matrix mineralisation (both plate and trabeculae) in relation to histomorphometric parameters of bone remodelling and osteocyte lacunae (OL) characteristics in the tibial plateau (TP) of KOA patients with and without BMLs (OA-BML and OA No-BML, respectively) in comparison to nonOA cadaveric controls (CTL). Osteochondral (cartilage-bone) tissue was sampled from the BML signal region within the medial compartment for each OA-BML TP, and from a corresponding medial region for OA No-BML and CTL TPs. The tissue samples were embedded in resin, and sections stained with Von-Kossa Haematoxylin and Eosin (H&E) for quantitation of static indices of bone remodelling. Resin blocks were then further polished, and carbon-coated for quantitative backscattered electron imaging (qBEI) to determine the bone mineralisation density distribution (BMDD), as well as OL characteristics. It was found that OA-BML contained higher osteoid volume per tissue volume (OV/TV; %) and per bone volume (OV/BV; %) in both subchondral plate and trabecular bone compared to OA No-BML and CTL. The BMDD of OA-BML in both subchondral plate and trabecular bone was shifted toward a lower degree of mineralisation. Typically, an increase in both the heterogeneity of mineralisation density (Ca Width; wt%Ca) and the percentage of lower calcium (Ca Low; % B.Ar) in trabecular bone with OA-BML versus CTL was observed. Further, unmineralised OL density (#/mm2) in subchondral plate was distinctly higher in OA-BML samples compared to CTL. The KOA patients with and without BMLs had significantly decreased density of mineralised OL (#/mm2) in trabecular bone compared to CTL. Taken together, these findings indicate that tibial BMLs in advanced KOA patients are characterised by significantly hypo-mineralised subchondral bone compared with CTL. These differences associated with evidence of increased bone remodelling in OA-BML, and may influence the mechanical properties of the subchondral bone, with implications for the overlying cartilage.

Gelatin-coated indium tin oxide slides improve human cartilage-bone tissue adherence and N-glycan signal intensity for mass spectrometry imaging.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has been successfully used to elucidate the relative abundance and spatial mapping of analytes in situ. Currently, sample preparation workflows for soft formalin-fixed paraffin-embedded (FFPE) tissues, such as brain, liver, kidney, and heart, have been successfully developed. However, hard tissues, such as cartilage-bone, tooth, and whole mouse body, have resulted in the loss of morphology or tissue during the heat-induced epitope retrieval (HIER) step on commercially available conductive indium tin oxide (ITO) slides. Therefore, we have successfully developed a novel and cost-effective sample preparation workflow in which commercial conductive ITO slides are pre-coated with gelatin and chromium potassium sulfate dodecahydrate to improve the adherence of FFPE human osteoarthritic cartilage-bone tissue sections. Gelatin-coated ITO slides also resulted in overall higher N-glycan signal intensity for not only FFPE osteoarthritic cartilage-bone tissue but also for FFPE hard-boiled egg white used as a quality control to assess the quality of sample preparation and MALDI-MSI acquisition. In summary, we present a novel straightforward workflow to improve slide adherence and morphological preservation of FFPE cartilage-bone tissue sections during HIER while improving the signal intensity of N-glycans spatially mapped from the same tissue sections by MALDI-MSI.

Novel technical developments in mass spectrometry imaging in 2020: A mini review.

The applicability of mass spectrometry imaging (MSI) has exponentially increased with the improvement of sample preparation, instrumentation (spatial resolution) and data analysis. The number of MSI publications listed in PubMed continues to grow with 378 published articles in 2020-2021. Initially, MSI was just sensitive enough to identify molecular features correlating with distinct tissue regions, similar to the resolution achieved by visual inspection after standard immunohistochemical staining. Although the spatial resolution was limited compared with other imaging modalities, the molecular intensity mapping added a new exciting capability. Over the past decade, significant improvements in every step of the workflow and most importantly in instrumentation were made, which now enables the molecular analysis at a cellular and even subcellular level. Here, we summarize the latest developments in MSI, with a focus on the latest approaches for tissue-based imaging described in 2020.

Mass Spectrometry Imaging as a Potential Tool to Investigate Human Osteoarthritis at the Tissue Level.

Osteoarthritis (OA) is the most common degenerative joint disease, predicted to increase in incidence year by year due to an ageing population. Due to the biological complexity of the disease, OA remains highly heterogeneous. Although much work has been undertaken in the past few years, underlying molecular mechanisms leading to joint tissue structural deterioration are not fully understood, with only few validated markers for disease diagnosis and progression being available. Discovery and quantitation of various OA-specific biomarkers is still largely focused on the bodily fluids which does not appear to be reliable and sensitive enough. However, with the advancement of spatial proteomic techniques, several novel peptides and proteins, as well as N-glycans, can be identified and localised in a reliable and sensitive manner. To summarise the important findings from OA biomarker studies, papers published between 2000 and 2020 were searched via Google Scholar and PubMed. Medical subject heading (MeSH) terms 'osteoarthritis', 'biomarker', 'synovial fluid', 'serum', 'urine', 'matrix-assisted laser desorption/ionisation', 'mass spectrometry imaging', 'proteomic', 'glycomic', 'cartilage', 'synovium' AND 'subchondral bone' were selectively used. The literature search was restricted to full-text original research articles and written only in English. Two main areas were reviewed for OA biomarker studies: (1) an overview of disease-specific markers detected from different types of OA bio-samples, and (2) an up-to-date summary of the tissue-specific OA studies that have utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). Overall, these OA biomarkers could provide clinicians with information for better the diagnosis, and prognosis of individual patients, and ultimately help facilitate the development of disease-modifying treatments.

Raman microspectroscopy demonstrates reduced mineralization of subchondral bone marrow lesions in knee osteoarthritis patients.

INTRODUCTION: Bone marrow lesions (BMLs) are frequently identified by MRI in the subchondral bone in knee osteoarthritis (KOA). BMLs are known to be closely associated with joint pain, loss of the cartilage and structural changes in the subchondral trabecular bone (SCTB). Despite this, understanding of the nature of BMLs at the trabecular tissue level is incomplete. Thus, we used Raman microspectroscopy to examine the biochemical properties of SCTB from KOA patients with presence or absence of BMLs (OA-BML, OA No-BML; respectively), in comparison with age-matched cadaveric non-symptomatic controls (Non-OA CTL). METHODS: Tibial plateau (TP) specimens were collected from 19 KOA arthroplasty patients (6-Male, 13-Female; aged 56-74 years). BMLs were identified ex-vivo by MRI, using PDFS- and T1-weighted sequences. The KOA specimens were then categorized into an OA-BML group (n = 12; containing a BML within the medial condyle only) and an OA No-BML group (n = 7; with no BMLs identified in the TP). The control (CTL) group consisted of Non-OA cadaveric TP samples with no BMLs and no macroscopic or microscopic evidence of OA-related changes (n = 8; 5-Male, 3-Female; aged 44-80 years). Confocal Raman microspectroscopy, with high spatial resolution, was used to quantify the biochemical properties of SCTB tissue of both the medial and the lateral condyle in each group. RESULTS: The ratios of peak intensity and integrated area of bone matrix mineral (Phosphate (v1), Phosphate (v2) and Phosphate (v4)), to surrogates of the organic phase of bone matrix (Amide I, Proline and Amide III), were calculated. Within the medial compartment, the mineral:organic matrix ratios were significantly lower for OA-BML, compared to Non-OA CTL. These ratios were also significantly lower for the OA-BML medial compartment, compared to the OA-BML lateral compartment. There were no group or compartmental differences for Carbonate:Phosphate (v1, v2 and v4), Amide III (α-helix):Amide III (random-coil), Hydroxyproline:Proline, or Crystallinity. CONCLUSION: As measured by Raman microspectroscopy, SCTB tissue in BML zones in KOA is significantly less mineralized than the corresponding zones in individuals without OA. These data are consistent with those obtained using other methods (e.g. Fourier transform infrared spectroscopy; FTIR) and with the increased rate of bone remodeling observed in BML zones. Reduced mineralization may change the biomechanical properties of the trabecular bone in BMLs and the mechanical interaction between subchondral bone and its overlying cartilage, with potential implications for the development and progression of OA.

Bone matrix microdamage and vascular changes characterize bone marrow lesions in the subchondral bone of knee osteoarthritis.

INTRODUCTION: Bone marrow lesions (BMLs) in the subchondral bone in osteoarthritis (OA) are suggested to be multifactorial, although the pathogenic mechanisms are unknown. Bone metabolism and cardiovascular risk factors associate with BML in epidemiologic studies. However, there are no studies at the tissue level investigating the relationship between these processes and BML. The aim of this study was to investigate the relationship between BMLs in the tibial plateau (TP) of knee OA and bone matrix microdamage, osteocyte density and vascular changes. METHODS: TP were obtained from 73 patients at total knee replacement surgery and BMLs were identified ex vivo in TP tissue using MRI. Comparator 'No BML' tissue was from matched anatomical sites to the BMLs. Quantitative assessment was made of subchondral bone microdamage, bone resorption indices, osteocyte cellularity, and vascular features. RESULTS: Several key parameters were different between BML and No BML tissue. These included increased microcrack burden (p = .01, p = .0001), which associated positively with bone resorption and negatively with cartilage volume, and greater osteocyte numerical density (p = .02, p = .01), in the subchondral bone plate and subchondral trabeculae, respectively. The marrow tissue within BML zones contained increased arteriolar density (p = .04, p = .0006), and altered vascular characteristics, in particular increased wall thickness (p = .007) and wall:lumen ratio (wall thickness over internal lumen area) (p = .001), compared with No BML bone. CONCLUSIONS: Increased bone matrix microdamage and altered vasculature in the subchondral bone of BMLs is consistent with overloading and vascular contributions to the formation of these lesions. Given the important role of BMLs in knee OA, these contributing factors offer potential targets for the treatment and prevention of knee OA.