Further improvements on instrumentation sensitivity, matrix selection, image processing and sample preparation will expand the application of MALDI-MSI in plant research.

Low-molecular-weight (low-MW) compounds have many essential functions in biological processes, and the molecular imaging of as many low-MW compounds as possible is critical for understanding complex biological processes. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an emerging molecular-imaging technology that enables determination of the spatial distributions and the relative abundances of diverse endogenous compounds in tissues. New matrices suitable for the imaging of low-MW compounds by MALDI-MSI are important for the technological advancement of tissue imaging. In this study, 3,4-dimethoxycinnamic acid (DMCA) was evaluated as a new matrix for enhanced low-MW compound detection by MALDI-MSI because of its strong ultraviolet absorption, low matrix-ion related interferences below m/z 500, and high ionization efficiency for the analysis of low-MW compounds. DMCA was successfully used for improved in situ detection of low-molecular-weight metabolites (m/z < 500) and lipids in rat liver, rat brain, and germinating Chinese-yew seed tissue sections. The use of DMCA led to the successful in situ detection of 303, 200, and 248 low-MW compound ion signals from these three tissues, respectively. Both MALDI-MS/MS and LC-MS/MS were used to identify these ion signals, leading to the identification of 115 low-MW compounds from rat liver (including 53 lipids, 29 oligopeptides, and 33 metabolites), 130 low-MW compounds from rat brain (including 104 lipids, 5 oligopeptides, and 21 metabolites), and 111 low-MW compounds from germinating Chinese-yew seeds (including 77 lipids, 22 oligopeptides, 8 flavonoids, and 4 alkaloids). A larger number of low-MW compounds could be detected and imaged when DMCA was used as the MALDI matrix than with other commonly used MALDI matrices such as 2,5-dihydroxybenzoic acid, α-cyano-4hydroxycinnamic acid, 2-mercaptobenzothiazole, graphene oxide, and silver nanoparticles. Our work provides a new and powerful matrix for enhanced MALDI-MS profiling of low-MW compounds in both animal and plant tissues.E ndogenous low-molecular-weight (low-MW) compounds, especially low-molecular-weight metabolites (m/z < 500) and lipids, perform multiple essential functions in multitudinous biological processes, including energy transformation,

To our knowledge, this was the first study in which caffeic acid (CA) was successfully evaluated as a matrix to enhance the in situ detection and imaging of endogenous proteins in three biological tissue sections (i.e., a rat brain and Capparis masaikai and germinating soybean seeds) by matrix-assisted laser desorption/ ionization mass spectrometry imaging (MALDI-MSI). Our results show several properties of CA, including strong ultraviolet absorption, a super-wide MS detection mass range close to 200,000 Da, micrometer-sized matrix crystals, uniform matrix deposition, and high ionization efficiency. More high-molecularweight (HMW) protein ion signals (m/z > 30,000) could be clearly detected in biological tissues with the use of CA, compared to two commonly used MALDI matrices, i.e., sinapinic acid (SA) and ferulic acid (FA). Notably, CA shows excellent performance for HMW protein in situ detection from biological tissues in the mass range m/z > 80,000, compared to the use of SA and FA. Furthermore, the use of a CA matrix also significantly enhanced the imaging of proteins on the surface of selected biological tissue sections. Three HMW protein ion signals (m/z 50,419, m/z 65,874, and m/z 191,872) from a rat brain, two sweet proteins (mabinlin-2 and mabinlin-4) from a Capparis masaikai seed, and three HMW protein ion signals (m/z 94,838, m/z 134,204, and m/z 198,738) from a germinating soybean seed were successfully imaged for the first time. Our study proves that CA has the potential to become a standard organic acid matrix for enhanced tissue imaging of HMW proteins by MALDI-MSI in both animal and plant tissues.

Previous studies showed that SDF-1α is a catabolic factor that can infiltrate cartilage, decrease proteoglycan content, and increase MMP-13 activity. Inhibiting the SDF-1α/CXCR4 signalling pathway can attenuate the pathogenesis of osteoarthritis (OA). Recent studies have also shown that SDF-1α enhances chondrocyte proliferation and maturation. These results appear to be contradictory. In the current study, we used a destabilisation OA animal model to investigate the effects of SDF-1α/CXCR4 signalling in the tibial subchondral bone and the OA pathological process. Post-traumatic osteoarthritis (PTOA) mice models were prepared by transecting the anterior cruciate ligament (ACLT), or a sham surgery was performed, in a total of 30 mice. Mice were treated with phosphate buffer saline (PBS) or AMD3100 (an inhibitor of CXCR4) and sacrificed at 30 days post ACLT or sham surgery. Tibial subchondral bone status was quantified by micro-computed tomography (μCT). Knee-joint histology was analysed to examine the articular cartilage and joint degeneration. The levels of SDF-1α and collagen type I c-telopeptidefragments (CTX-I) were quantified by ELISA. Bone marrow mononuclear cells (BMMCs) were used to clarify the effects of SDF-1α on osteoclast formation and activity in vivo. μCT analysis revealed significant loss of trabecular bone from tibial subchondral bone post-ACLT, which was effectively prevented by AMD3100. AMD3100 could partially prevent bone loss and articular cartilage degeneration. Serum biomarkers revealed an increase in SDF-1α and bone resorption, which were also reduced by AMD3100. SDF-1α can promote osteoclast formation and the expression oftartrate resistant acid phosphatase (TRAP), cathepsin K (CK), and matrix metalloproteinase (MMP)-9 in osteoclasts by activating the MAPK pathway, including ERK and p38, but not JNK. In conclusion, inhibition of SDF-1α/CXCR4signalling was able to prevent trabecular bone loss and attenuated cartilage degeneration in PTOA mice.