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The proteomic profile of the human myotendinous junction

Summary Proteomics analysis of skeletal muscle has recently progressed from whole muscle tissue to single myofibers. Here, we further focus on a specific myofiber domain crucial for force transmission from muscle to tendon, the myotendinous junction (MTJ). To overcome the anatomical constraints preventing the isolation of pure MTJs, we performed in-depth analysis of the MTJ by progressive removal of the muscle component in semitendinosus muscle-tendon samples. Using detergents with increasing stringency, we quantified >3000 proteins across all samples, and identified 112 significantly enriched MTJ proteins, including 24 known MTJ-enriched proteins. Of the 88 novel MTJ markers, immunofluorescence analysis confirmed the presence of tetraspanin-24 (CD151), kindlin-2 (FERMT2), cartilage intermediate layer protein 1 (CILP), and integrin-alpha10 (ITGA10), at the human MTJ. Together, these human data constitute the first detailed MTJ proteomics resource that will contribute to advance understanding of the biology of the MTJ and its failure in pathological conditions.


INTRODUCTION
A major progress in the definition of the protein profile of skeletal muscle, made possible by recent developments in liquid chromatography-mass spectrometry (LCMS)-based approaches, has been the transition from bulk muscle  to single fiber proteomics (Murgia et al., , 2017(Murgia et al., , 2021Deshmukh et al., 2021;Kallabis et al., 2020;Lang et al., 2018). Here, we take a step further in this direction and report the first detailed proteomic analysis of a specific myofiber domain, the myotendinous junction (MTJ). The MTJ is responsible for the transmission of contractile force from muscle to skeleton; thus, knowledge of its molecular composition is essential to understand the process of mechanotransduction at the muscletendon interface and its failure under pathological conditions. The MTJ is highly specialized in both structure and composition to meet the challenges of linking two very different tissues and withstand great mechanical stresses. Structural specialization of the MTJ includes finger-like processes, where collagen fibrils from the tendon interdigitate with extensive folding/invagination in the muscle fiber membrane at the tips of the muscle fibers. These finger-like processes have been estimated to increase the surface contact area between the muscle fibers and tendon by up to 20-fold (Knudsen et al., 2015), thereby reducing the stress, and increasing the strength, at the interface between muscle fibers and tendon (Kojima et al., 2008;Tidball, 1991). Despite the high degree of specialization, the MTJ is the main site of muscle strain injuries. Previous strain injuries, together with aging, are the strongest risk factors for strain injury (Green et al., 2020). Moreover, approximately four out of 5 re-injuries occur at the same site as the original injury (Wangensteen et al., 2016), indicating suboptimal healing of the MTJ after injury.
Despite this important role in injuries, our knowledge of the MTJ composition in general, and the human MTJ in particular, remains limited, mainly because access to human MTJ is restricted to surgically removed tissue. Moreover, the study of the MTJ has been limited to immunohistochemical studies that showed the enrichment of different sarcolemmal proteins at the myofiber tip, which is not surprising given the increased surface area at these sites. The most important finding has been the discovery that collagen XXII is specifically located at the MTJs (Koch et al., 2004). Recently, single-nucleus RNA-seq studies of mouse muscles have identified a specific cluster of MTJ myonuclei with a distinct mRNA profile, including COL22A1 mRNA, supporting the notion of a specialized transcriptional domain at the myofiber tip (Chemello et al., 2020;Dos Santos et al., 2020;Kim et al., 2020;Petrany et al., 2020;Wen et al., 2021). However, an unbiased inventory of the proteins detected at the MTJ based on proteomics analyses is not yet available.
Anatomical constraints do not allow for isolation of pure MTJ from muscle and tendon. Moreover, the proteomic analysis of these specialized tissues is challenging due to their structure and composition Sato et al., 2016). Two previous studies have attempted to map MTJ-specific proteins in the rodent muscle. However, the coverage of the proteome was incomplete in one study (Can et al., 2014), in which collagen XXII was not even identified, and the other study was limited to the analysis of extracellular matrix components (Jacobson et al., 2020). Here, we devised an enrichment approach by progressively reducing the muscle component of human semitendinosus muscle/tendon samples and extracting proteins through gradual increase in the stringency of the detergents. Each protein fraction was subsequently analyzed by MS-based proteomics, allowing mapping of the first human MTJ proteome. It is expected that these data will provide a resource and the basis for a better understanding of the intact healthy human MTJ and its remodeling under pathological conditions.

Sequential protein extraction allows for a deep muscle and tendon proteome
Samples of intact human semitendinosus muscle-tendon complex were collected from four individuals during anterior cruciate ligament (ACL) reconstruction surgery and divided a priori into three sample types by progressively scraping the muscle tissue off the tendon: i) non-scraped samples, consisting of muscle and tendon (muscle/tendon, M/T), ii) samples with the muscle partially removed to obtain less muscle on the tendon (a little muscle with tendon, (M)/T), and iii) samples with all visible muscle scraped away (MTJ) (Figure 1A). In addition, four human patellar tendon biopsies (T), which are not anatomically connected to skeletal muscle tissue, were included as a pure tendon control. The use of buffers with increasing detergent concentration allowed for the sequential extraction of proteins in M/T, (M)/T, MTJ, and T samples, which were then prepared for LCMS-based proteomics ( Figure 1A). We first analyzed the individual samples from the sequential protein extractions as independent experiments. This allowed for the study of the accommodation of this method to investigate MTJ proteins (Table S1) as it showed the segregation by extraction buffer (Figure S1A) or sample type ( Figure S1B). We observed the highest abundance of the MTJ-marker collagen XXII in the MTJ samples (Table S1). Moreover, Pearson correlation analysis and hierarchical clustering analysis revealed that the (M)/T samples were closely related to M/T and MTJ samples (Figures S1C and S1D). Hence, the intermediate (M)/T sample was removed from the further analysis. Expectedly, removal of the intermediate (M)/T sample resulted in a better segregation between the sample types ( Figure S1E). Of note, this segregation was not explained by inter-individual variability among participants ( Figure S1F), and the number of proteins detected in each extraction fraction was similar across samples types ( Figure S1G).
For the statistical comparison, after removal of the (M)/T sample, proteins quantified in different extraction fractions were combined into their respective sample type. We obtained comprehensive proteome coverage, quantifying 3918 proteins across all samples and on an average > 3000 proteins in each sample type (Table S2). The quantified proteome spanned across six orders of magnitude and included low-abundant proteins such as cytokines (interleukin 33: IL33; C-C-motif chemokine ligand 14 and 18: CCL14 and CCL18), kinases (phosphatidylinositol 4-kinase type 2 alpha: PI4K2A; mechanistic target of rapamycin kinase: MTOR, epidermal growth factor receptor: EGFR), transcription factors (nuclear factor kappa B subunit 1: NFKB1, BCL2-associated transcriptor factor 1: BCLAF1), as well as 121 extracellular matrix (ECM) proteins belonging to the core matrisome (21 collagens, 83 ECM glycoproteins, and 17 proteoglycans) (Naba et al., 2012), which are generally difficult to detect due to their lower solubility (Table S2). In this analysis, we quantified 2789, 2963, and 2584 proteins in M/T, MTJ, and T, respectively (Table S2, Figure 1B). The vast majority of proteins (2172) were quantified in all three sample types in at least three out of four subjects ( Figure 1C). A principal component analysis (PCA) showed a clear segregation of each sample type (Figure 1D), and a low and comparable inter-individual variance between subject within each sample type. The segregation of the sample types was driven by different proteins ( Figure 1E, in red), such as collagen I and XIRP2 (xin actin-binding repeat-containing protein 2/xinb), specific proteins for tendon and MTJ, respectively. Additionally, we performed hierarchical clustering of the proteome of the three sample types ( Figure 1F), and observed i) the samples separated clearly, with MTJ being closer to tendon (T) than the muscle/tendon (M/T) and ii) three clear expression patterns: a cluster of proteins increased only in MTJ iScience Article samples ( Figure 1F, salmon color), increased in MTJ and T (light blue) and increased only in M/T (golden brown). We then performed the enrichment analysis using GOCC (Gene Ontology Cellular Component) terms within these clusters in order to assess the cellular localization of the majority of proteins (Table S3). Proteins belonging to the sarcoglycan complex were enriched in the MTJ cluster ( Figure 1F, salmon); proteins associated with collagen and located in the ECM were over-represented among the proteins increased in the MTJ and T sample types ( Figure 1F, light blue), while proteins related to the muscle myosin complex and mitochondria were enriched in the M/T samples ( Figure 1F, golden brown). Details regarding the other enriched protein categories for individual clusters can be found in Table S3.

Proteomics analysis revealed known and novel proteins enriched at the MTJ region
To identify proteins enriched at the MTJ region, we performed pairwise two-samples t test between the proteins in the MTJ vs M/T and MTJ vs T samples (Figures 2A and 2B). In the comparison between MTJ and M/T, 1156 (36.4%) proteins were significantly increased in MTJ (Figure 2A, red, Table S2), whereas in the comparison between MTJ and T, 665 (21.4%) proteins returned which were significantly increased in MTJ ( Figure 2B, red, Table S2).
These results were then combined to identify proteins significantly increased in MTJ in both pair comparisons, thus enriched in the MTJ samples, resulting in a final dataset with a total of 112 proteins (Figures 2C and 2D, in red, Table S4). Of these, we can report 88 novel MTJ-enriched proteins, together with 24 proteins known to be enriched at the MTJ in vertebrates (Table S4), including collagen XXII (COL22A1), which is exclusively confined to the MTJ in the skeletal muscle-tendon unit (Jakobsen et al., 2017;Koch et al., 2004), as well as several laminin subunits and 10 proteins belonging to the dystrophin glycoprotein complex (DGC) which is known to be enriched at the MTJ (Charvet et al., 2012;Grady et al., 2003).
To gain further insights into the 112 MTJ-specific proteins, we performed an enrichment analysis within these proteins using GOCC and UniProt Keywords annotations ( Figure 2E and Table S5), which revealed an over-representation of cell membrane-related annotations. The two most enriched GOCC terms were the sarcoglycan and the laminin complexes (enrichment factors: 21.3 and 17.8, respectively), and the two most enriched Uniprot Keywords were ''lamininEGF-likedomain'' (enrichment factor: 13.3) and ''GPI-anchor'' (enrichment factor: 10.3).

Confirmatory validation of the isolation of the MTJ region with known MTJ-enriched proteins
We then performed immunofluorescent staining of selected proteins significantly enriched at the MTJ versus M/T and T, and known to be present in various cellular compartments of the MTJ in vertebrate (XIRP2, thrombospondin-4 (THBS4)) or human (neural cell adhesion molecule 1 (NCAM1)) ( Figure 3) to validate the MS-based proteomic data. For illustrative purposes Figure 3A is a representative image of a 10-mm thick MTJ section of human semitendinosus, cut in the longitudinal plane of the muscle fibers, and stained for myosin heavy chain, collagen XXII, and tenascin C, to visualize the muscle fibers, MTJ, and tendon, respectively. A very strong immunofluorescent signal for XIRP2 was evident ( Figure 3B), exclusively confined to the MTJ on the muscle fiber side of the collagen XXII staining. In contrast to XIRP2, THBS4 was enriched, but not exclusively confined to the MTJ ( Figure 3C), as THBS4 signal was also seen in the tendon together with a vague signal outside the dystrophin-stained sarcolemma along the muscle fibers ( Figure 3C). NCAM1 was the only MTJ-enriched protein in the proteome dataset identified from razor peptides only (Table S2), and the immunofluorescent staining showed a concentrated signal in the muscle fiber cytoplasm for a length of 200-300 mm before fiber termination at the MTJ ( Figure 3D).

Validation of novel MTJ-enriched proteins
MS-based proteomics and a stringent downstream bioinformatics analysis led to discovery of 88 novel MTJ-enriched proteins, which we aimed to confirm as MTJ-enriched by immunofluorescence ( Figure 4). Tetraspanin-24 (CD151/PETA-3, Figure 4A) and kindlin-2 (FERMT2/UNC112B/MIG2, Figure 4B) showed very strong staining exclusively at the MTJ, on the muscle side of collagen XXII, and the laminin-stained basement membrane. A weak tetraspanin-24 signal could also be observed along the muscle fiber membrane in some fibers ( Figure 4A). Similarly, cartilage intermediate layer protein 1 (CILP) staining was strong at the muscle fiber tips at the muscle side of collagen XXII and in close proximity to the dystrophin-stained sarcolemma, while a weak CILP reactivity was also seen in the muscle fiber cytoplasm, which appeared stronger in few of the fibers ( Figure 4C). In addition, a positive signal was seen for integrin-a10 (ITGA10) iScience Article at the muscle fiber tips in close proximity to collagen XXII ( Figure 4D), although at a lower intensity compared to collagen XXII, XIRP2, tetraspanin-24, kindlin-2, and CILP.

DISCUSSION
A major recent advance in the proteomic analysis of skeletal muscle has been the development of procedures for single-fiber proteomics. Here, we have zoomed in even further on the MTJ, the specialized domain iScience Article at the muscle-tendon interface. The MTJ is structurally organized in a complex manner and cannot be anatomically isolated from muscle and tendon, posing a challenge for MS-based proteomics analysis due to presence of high-abundant proteins Sato et al., 2016). Moreover, the MTJ consists of proteins belonging to the ECM, which represents a cross-linked and insoluble assembled matrix. It is therefore challenging to extract the proteins from this tissue to study the proteomic composition of the MTJ. To overcome these challenges, we accommodated an experimental approach including scraping the semitendinosus tendon free of muscle tissue before using buffers with increasing stringency to obtain samples enriched in MTJ proteins. The extraction procedure, based on progressively increasing detergent concentration, was essentially similar to that applied by (Schiller et al., 2015) to characterize lung fibrosis. We identified 112 MTJ-enriched proteins of which 24 were known and 88 were novel. Using immunofluorescence, we further confirmed enrichment of the novel MTJ-specific tetraspanin-24, kindlin-2, CILP, and integrin-a10, together representing pathways in force transmission, membrane folding, and extracellular matrix binding. These results represent the first MS-based proteome of the human MTJ and will serve as a valuable resource for developing methods for proteomic studies of specialized domains and the understanding of adaptation and failure of force transmission and regeneration at the MTJ.
The tips of the muscle fibers, where they insert onto the tendon at the MTJ, represent a distinct myocellular domain, characterized by the expression of certain proteins such as Nestin (Vaittinen et al., 1999) and NCAM1 (Daniloff et al., 1989;Jakobsen et al., 2018), not expressed in the rest of the musculotendinous unit. As the muscle fibers (which can be several centimeters long) approach the MTJ, collagen XXII is observed in the muscle fiber basement membrane (Koch et al., 2004). We initially compared semitendinosus muscle-tendon samples with decreasing amounts of muscle by gradually scraping the muscle off the tendon to varying degrees. The greatest enrichment of collagen XXII was detected in the samples that had been most harshly scraped, presumably because the tips of the muscle fibers are deeply embedded in the tendon and the MTJ-enriched proteins are not masked by an excess of muscle proteins. This observation formed the basis of the methodology applied in the present study, where we also included a biopsy of pure patellar tendon (which is not anatomically connected to skeletal muscle and is therefore completely free of muscle-and MTJ-specific proteins) to identify tendon-specific proteins in the MTJ samples. Furthermore, our methodological approach included a sequential protein extraction with buffers of increasing detergent concentration before MS-based proteomics, in combination with a downstream stringent filter that included only those proteins quantified in at least three out of four subjects (biological replicates).
Our methodological approach was validated by an enrichment analysis within the 112 MTJ-enriched proteins that revealed an over-representation of GOCC and UniProt Keywords annotation terms related to ''sarcoglycan complex'', ''laminin complex'', ''cell junction'', ''membrane'', ''adhesion'', and ''extracellular matrix''. These annotations verified that proteins in our list can be located in both the muscle and the tendon side of the MTJ. The high prevalence of proteins belonging to the DGC in the MTJ is not surprising given that DGC proteins are known to be more abundant in the MTJ domain of the myofibers (Charvet et al., 2012;Grady et al., 2003). Moreover, these proteins may have contributed to many of the enriched GOCC terms and UniProt Keywords. Altogether, these enriched annotations reflect that the MTJ is rich in membrane components, in line with the finger-like protrusions that form extensive membrane foldings at the tip of the muscle fibers (Knudsen et al., 2015). The enrichment of annotations such as ''cell junction'', ''adherens junction'', and ''anchored to membrane'' further reflect the MTJ-specific requirement of forming a strong link between muscle fibers and tendon.
Several of the 112 MTJ-enriched proteins belonged to the extracellular matrisome (Naba et al., 2012) and have not previously been associated with the MTJ, including but not limited to: Agrin (AGRN), vitrin (VIT), metalloproteinase inhibitor 3 (TIMP3), annexin A3 and A4 (ANXA3, ANXA4), plexin-A1 (PLXNA1), and syndecan-2 (SDC2). Notably, annexin A1 was observed in mouse MTJ with proteomics and immunofluorescence (Can et al., 2014), while our data suggest a role for other members of the annexin family at the human iScience Article MTJ. Several other novel and potentially interesting MTJ-enriched proteins were observed, such as anthrax toxin receptor 1 (ANTXR1), a transmembrane protein shown to interact with actin and affect cell adhesion; caveolin (CAV2), involved in formation of invaginations to the plasma membrane; the actin binding coactosin-like protein (COTL1); the ephrin type-A receptor 2 (EPHA2), involved in integrin-mediated adhesion and formation of tissue boundaries during embryogenesis; beta-parvin (PARVB), an actin-binding protein with a role in cytoskeleton organization and cell adhesion; and reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored matrix metalloproteinase-regulator also found in the NMJ (Kawashima et al., 2008). In addition, we also observed enrichment of hemicentin-1 (HMCN1) in MTJ, which is known to link tissues together at the basement membrane and was very recently described at the MTJ (Welcker et al., 2021). The list also contained several proteins located in the Golgi apparatus and/or ER or involved in processes related to endocytosis, autophagy, microtubules, T-cells, enzymatic activity, and the nucleus/transcriptional machinery, together expanding insight into the complex diversity in proteomic composition and cellular processes at the MTJ.
By using immunofluorescence, we confirmed the presence of known components of the MTJ (i.e., XIRP2, THBS4, and NCAM1). XIRP2 and THBS4 have not previously been shown in human MTJ, and the positive immunofluorescent staining of XIRP2, THBS4, and NCAM1 at the MTJ region ( Figures 3B-3D) validates our method to detect MTJ-enriched proteins with distinct staining profiles and enrichment scores.
We then performed immunofluorescent stainings for selected targets among the 88 MTJ-enriched proteins in our dataset, which have not been previously reported in the myotendinous location. Kindlin-2 and tetraspanin-24 are both known to associate with the transmembrane integrins and were therefore chosen for further examination by immunofluorescence. Similarly to XIRP2, stainings of tetraspanin-24 (Sterk et al., 2002) and kindlin-2 (Dowling et al., 2008) have been shown at the intercalated disc of the human and murine heart, respectively, as well as at the costameres in cardiac muscle cells and skeletal muscle fibers (Dowling et al., 2008;Sterk et al., 2002). To our knowledge, this is the first report of tetraspanin-24 and kindlin-2 enrichment at the MTJ in skeletal muscle fibers. Although the specific role of these proteins at the MTJ is currently unknown, we find it relevant that tetraspanins are involved in both membrane invagination (Yue et al., 2017) and linking cell membranes via morphogenesis of microextrusions (Huang et al., 2018), whereas kindlin-2 plays a key role in the induction of membrane protrusions and formation of lamellipodia during early isotropic fibroblast cell spreading (Theodosiou et al., 2016). Altogether, this could indicate a role for tetraspanin-24 and kindlin-2 in the extensive folding/invaginations of the tendon into the muscle fiber membrane at the MTJ region.
Some of the novel MTJ-enriched proteins are traditionally associated with cartilage. Given that the MTJmarker collagen XXII has also been identified in the articular surface of cartilage (Koch et al., 2004), we decided to further examine the location of two cartilage-associated proteins at the MTJ: the integrin-a10 subunit that is expressed together with the b1-subunit in cartilage chondrocytes (Camper et al., 1998) and the cartilage intermediate layer protein 1 (CILP), which is located in the mid-zone of human articular cartilage (Lorenzo et al., 1998). Integrin-a10b1 belongs to a group of collagen-binding integrins (a1b1, a2b1, a10b1, and a11b1), and collagen XXII shows binding affinity for all of them (Zwolanek et al., 2014). At this point, it is not fully clear which muscle fiber membrane proteins collagen XXII binds to at the MTJ, and our data could suggest a role for integrin-a10 as a binding partner for collagen XXII in adult human MTJ.
The localization of CILP near the sarcolemma at the MTJ ( Figure 4D) suggests that MTJ-specialized myonuclei may be the source of CILP. In line with this, recent snRNA-seq data in mice showed that CILP gene expression was increased in a sub cluster of MTJ myonuclei (Kim et al., 2020), but also in tenocytes (Petrany et al., 2020) and fibro-adipogenic precursors (Dos Santos et al., 2020). CILP was first discovered in cartilage as an extracellular matrix glycoprotein that increases with aging, progression of osteoarthritis (Lorenzo et al., 1998), and degeneration of intervertebral discs at the lumbar spine (Seki et al., 2005). At the protein level, CILP also shows immunoreactivity in fibrotic regions in human hearts from patients with ischemic cardiomyopathy, but not in healthy controls (Barallobre-Barreiro et al., 2012;Park et al., 2020). As a negative regulator of cardiac fibrosis, it is tempting to speculate that CILP also has an anti-fibrotic function in the MTJ of skeletal muscle, which could be important in a region that is subjected to repeated stress during daily muscle contractions, and also in the accumulation of fibrotic scar tissue after muscle strain injuries (Silder et al., 2008).
In addition to CILP, the pre-mRNAs coding for 19 out of the 112 MTJ-enriched proteins identified in human biopsies were selectively upregulated in MTJ-myonuclei clusters described in four recent snRNA-seq iScience Article studies of mouse skeletal muscle (Petrany et al., 2020;Dos Santos et al., 2020;Kim et al., 2020;Wen et al., 2021). Only COL22A1, XIRP2, LAMA2, and LAMC1 were found in all four studies; whereas NRAP and APP were found in three studies (Petrany et al., 2020;Dos Santos et al., 2020;Wen et al., 2021); ANXA4, ITGA7, SGCD, and SLC44A2 in two studies (Petrany et al., 2020;Wen et al., 2021); and several transcripts were found in one study: CILP, ITIH5, LAMB1 (Kim et al., 2020); DES, SORBS1 (Dos Santos et al., 2020); FERMT2, LAMB2, NCAM1 (Wen et al., 2021); RRAS2, SGCG (Petrany et al., 2020). Along with CILP, these 19 proteins constitute both known and novel MTJ-enriched proteins. Furthermore, in snRNA-seq data from Ex51 mice (a mouse model of Duchenne's muscular dystrophy) (Chemello et al., 2020), the gene transcripts for 26 of the 112 MTJ-enriched proteins were differentially regulated (15 upregulated and 11 downregulated). The observation that many of the 112 MTJ-enriched proteins reported here were not found in the snRNA-seq datasets may reflect a species difference (human vs. mouse) and the fact that many proteins at the MTJ do not necessarily originate from MTJ myonuclei, which underlines the specificity and value of measurements at the transcript and protein levels.
To our knowledge, only two studies have pioneered the study of the MTJ proteome in detail (Can et al., 2014;Jacobson et al., 2020). Can et al. used laser capture microdissection of MTJ and pure muscle, but not the tendon, from hindlimb and forelimb muscles in mice. While this approach should give a high enrichment of MTJ-specific proteins, our study demonstrates the limitations of not including a tendon control sample. Another limitation to this time-consuming method was the low total amount of isolated tissue, leading to the need to pool the samples from all biological replicates prior to LCMS analysis and impeding statistical analysis (Can et al., 2014). A different approach was used by Jacobson et al., in which the whole mouse soleus muscle was divided into pure muscle, pure tendon, and a muscle-tendon region that included the MTJ (Jacobson et al., 2020). In that study, the authors used buffers of increasing stringency to extract the insoluble proteins from the ECM and tendon prior to the MS analysis, similarly to the present study. However, our study was adapted to further improve the MTJ proteome coverage by performing an experimental approach where muscle tissue was scraped away to specifically isolate MTJ-enriched region differently than previous studies (Jacobson et al., 2020), therefore avoiding the masking of low-abundant MTJ-specific proteins by high-abundant muscle proteins. Altogether, in addition to the largest MTJ-enriched proteome, we generated the deepest human tendon proteome to date, as well as one of the most comprehensive human muscle proteome (Gonzalez-Freire et al., 2017) which could serve as a valuable resource for the scientific community.
The present study provides the first human MTJ proteome and a deeper understanding of the molecular components of this highly specialized myofiber domain. We combined graded muscle removal to enrich for proteins in the MTJ region, buffers with increasing stringency, and high-resolution MS-based proteomics to deliver the first human MTJ proteome to date. By using this methodology, we obtained a purer MTJ and quantified known MTJ-enriched proteins (i.e. collagen XXII, NCAM), thus demonstrating its fit to also overcome the high dynamic range that could mask low-abundant proteins in this region. The 88 novel MTJ marker proteins identified underline the high degree of specialization of this myofiber domain and, considering their functions in other tissues, suggest an important role in strengthening and repair of the MTJ. Overall, this study provides a valuable proteomics resource with novel insights into the composition of a myofiber domain that is highly specialized to withstand great mechanical forces and, paradoxically, also susceptible to strain injury rupture.

Limitations of the study
One of the limitations of this study is the challenge in obtaining ''pure'' MTJ tissue, because the MTJ is a combination of muscle fibers and their associated connective tissue on one side, and tendon tissue on the other. While the strong representation of known MTJ proteins in our MTJ fraction validates our approach of progressively removing the muscle tissue, it is likely that the method can be refined further. Remaining work also includes reconciling differences between the MTJ proteins identified in our study and the MTJ genes identified at the transcript level in the mouse.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: Human tissue was collected from eight healthy young males (20-44 years old), undergoing either anterior cruciate ligament (ACL) reconstruction surgery (n = 4) or patellar tendon biopsies (n = 4). Uninjured semitendinosus muscle-tendon tissue was collected during ACL surgeries following standard procedures. The surgeon harvested the semitendinosus tendon and secured the tissue needed for the ACL autograft before the excess (most proximal part) of the harvested tissue was placed in PBS in a falcon tube on ice and transferred to the laboratory. The sample consisted of bundles of muscle fibres attached to the semitendinosus tendon and was divided into three parts; M/T, comprised of unaltered muscle-tendon tissue; (M)/T was scraped gently with a scalpel to remove some, but not all, of the muscle from the tendon; MTJ was strongly scraped of all visible muscle tissue to obtain an enrichment of MTJ-proteins together with the tendon. Patellar tendon biopsies (T) were taken with a 14-G needle (Bard Magnum Biopsy Instrument, C.R. Bard, Covington, GA) under local anesthetics (lidocaine, 1%) after sterilization of the skin. All samples for proteomics were placed in cryotubes, frozen in liquid nitrogen and stored at À80 C. In addition, semitendinosus muscle-tendon tissue for histology was carefully aligned and embedded in Tissue-Tek, and frozen in isopentane pre-cooled in liquid nitrogen before storage in cryotubes at À80 C. 10 mm thick sections from human semitendinosus muscle-tendon samples were oriented in the longitudinal plane of the muscle fibers and cut in a cryostat at À20 C. The sections were collected on glass slides and stored at À80 C.
For the LCMSMS analysis, proteins (50 mg protein/sample, or all the sample if less) were digested using insolution digestion protocol. Samples were reduced with 0.1 M DTT for 30 min, and alkylated with 55 mM of IAA for 20 min in the dark, both steps taking place at room temperature. For the digestion, samples were digested using endoproteinase LysC (1 mg enzyme/50 mg protein) for 1 h at room temperature. The samples ll OPEN ACCESS

Computational MSMS data analysis
Mass spectra were analyzed using MaxQuant (version À1.6.01) with the integrated Adromeda search engine (Tyanova et al., 2016a). The initial maximum allowed mass deviation was set to 6 ppm for monoisotopic precursor ions and 20 ppm for MS/MS peaks. Enzyme specificity was set to trypsin, defined as C-terminal to arginine and lysine excluding proline, and a maximum of two missed cleavages were allowed. A minimal peptide length of six amino acids was required. Carbamidomethylcysteine was set as a fixed modification, while N-terminal acetylation and methio-nine oxidation were set as variable modifications. The spectra were searched by the Andromeda search engine against the Human UniProt sequence database combined with 248 common contaminants and concatenated with the reversed versions of all sequences. The false discovery rate (FDR) was set to 1% for peptide and protein identifications. To match the identifications across different LC-MS runs, the ''match between runs'' option in MaxQuant was enabled with a retention time window of 30 s. In the case of identified peptides that were shared between two or more proteins, these were combined and reported in protein group. Label free protein quantification was performed ll OPEN ACCESS iScience 25, 103836, February 18, 2022