Jie Gao, Yucun Zhang, Rui Zhu, Mu Li, Fei Xie, Changyin Li, Bingqian Li, Yungang Zhang
{"title":"Optical Sensing System Based on Bidirectional Self-Correction Technology: An Online Detection Method for Carbon Disulfide and Sulfur Dioxide in Gas-Insulated Switchgear","authors":"Jie Gao, Yucun Zhang, Rui Zhu, Mu Li, Fei Xie, Changyin Li, Bingqian Li, Yungang Zhang","doi":"10.1021/acs.analchem.5c00807","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00807","url":null,"abstract":"Carbon disulfide (CS<sub>2</sub>) and sulfur dioxide (SO<sub>2</sub>) are typical indicative gases for the early warning and diagnosis of faults in gas-insulated switchgear. In this study, an optical sensing system is reported for online detection of CS<sub>2</sub> and SO<sub>2</sub> based on a bidirectional self-correction technology (BSCT). First, the differential absorption signals of CS<sub>2</sub> and SO<sub>2</sub> in the wavelength range of 195–230 nm are obtained using the UV differential optical absorption spectroscopy (UV-DOAS) technique. On this basis, a BSCT is proposed to decouple the spectral lines that exhibit significant overlap. This method primarily employs bidirectional difference spectroscopy to mutually correct the spectra of CS<sub>2</sub> and SO<sub>2</sub>, combined with spectral reconstruction to extract the single-component absorption signals of CS<sub>2</sub> and SO<sub>2</sub> from the mixed gas spectra. Furthermore, the effectiveness of this novel decoupling technique is validated by comparing the decoupling results with the absorption spectra of single-component standard gases at corresponding concentrations. Finally, the quantitative relationships between the concentrations of target gases (CS<sub>2</sub>, SO<sub>2</sub>) and the optical parameters are modeled using the least-squares method. The experimental results show that the mean absolute percentage errors of CS<sub>2</sub> (19.00–3735.35 ppb) and SO<sub>2</sub> (0.19–38.77 ppm) are 0.543 and 0.521%, respectively. At an effective optical range of 50 cm, the system achieves the lowest detection limits of 0.5 ppb for CS<sub>2</sub> and 12 ppb for SO<sub>2</sub>, representing the best results reported to date for the online detection of CS<sub>2</sub> and SO<sub>2</sub> in the ppb-ppm range.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"72 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734224","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Juan Song, Yue Zheng, Xiaodan Huang, Xiyuan Yu, Rui Su, Zhi Zhu, Chaoyong Yang, Zengpeng Li, Yu Jiang, Huimin Zhang
{"title":"Enhancing Thermodynamic and Kinetic Performance of Microfluidic Interface-Based Circulating Fetal Cell Isolation for Noninvasive Prenatal Testing","authors":"Juan Song, Yue Zheng, Xiaodan Huang, Xiyuan Yu, Rui Su, Zhi Zhu, Chaoyong Yang, Zengpeng Li, Yu Jiang, Huimin Zhang","doi":"10.1021/acs.analchem.5c00711","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00711","url":null,"abstract":"Multivalent strategies have been widely applied in the microfluidic interface to boost the capture efficiency of target cells. However, achieving a balance between binding kinetics and thermodynamics in existing multivalent affinity interfaces remains challenging. Here, we propose a synergistic <b>A</b>ptamer-nanobody hetero-<b>M</b>ultivalency <b>P</b>rogrammable magnetic fluid microfluidic chip (<b>AMP-chip</b>) which utilizes the combined advantages of ligands to enhance both thermodynamic and kinetic properties of the capture interface. The AMP-chip integrates two distinct noninterfering recognition molecules: one with high affinity and another with rapid binding capability, both of which are assembled onto nanomagnetic beads. This integration achieves intermolecular complementarity, effectively enhancing the binding kinetics and thermodynamic stability. We chose mutually noninterfering CD71 recognition targets, a high-affinity nanobody (NB) and a rapid-binding aptamer (XQ 2d), and fully utilized the respective advantages of these ligands to facilitate rapid and tight recognition of the CD71 receptor on target cells. By integrating a herringbone microarray into an AMP-chip to further increase the cell–ligand interaction, we significantly improved the sensitivity and accuracy of circulating nucleated red blood cell (cNRBC) isolation from the peripheral blood mononuclear cells (PBMCs) of pregnant women. Additionally, the ligands were primarily fixed to the chip by magnetic force without chemical bonding, enabling nondestructive cell release and preserving high cell viability for subsequent molecular analyses. Overall, this approach offers a novel thermodynamic–kinetic synergistic heteromultivalency interface with significant potential for clinical applications.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"183 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ondřej Peterka, Yasmin Kadyrbekova, Robert Jirásko, Zuzana Lásko, Bohuslav Melichar, Michal Holčapek
{"title":"Novel Charge-Switch Derivatization Method Using 3-(Chlorosulfonyl)benzoic Acid for Sensitive RP-UHPLC/MS/MS Analysis of Acylglycerols, Sterols, and Prenols","authors":"Ondřej Peterka, Yasmin Kadyrbekova, Robert Jirásko, Zuzana Lásko, Bohuslav Melichar, Michal Holčapek","doi":"10.1021/acs.analchem.4c06496","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06496","url":null,"abstract":"Chemical derivatization involves the reaction of an analyte with a derivatization agent to modify its structure, improving the peak shape, chromatographic performance, structural analysis, ionization efficiency, and sensitivity. A novel derivatization method using 3-(chlorosulfonyl)benzoic acid is developed for the determination of monoacylglycerols, diacylglycerols, free sterols, and tocopherols using the reversed-phase ultra-high-performance liquid chromatography–tandem mass spectrometry (RP-UHPLC/MS/MS) method in the negative ion mode. The chromatographic and mass spectrometric properties of derivatized lipids are investigated by using 29 lipid standards spanning four lipid classes. The derivatization process is optimized using pooled plasma spiked by 9 internal standards, achieving an optimal yield with a reaction time of 40 min at 60 °C. The stability of the derivatives is confirmed, with short-term stability maintained for 10 h at 4 °C and long-term stability preserved for 5 days at −80 °C. The repeatability and reproducibility are verified by one/two operator(s), which underscores the simplicity and robustness of the method, and calibration curves with high linear regression coefficients illustrate the accuracy of the method. The derivatization approach, which combines RP-UHPLC/MS/MS and the use of specific fragmentation patterns, significantly reduces limits of detection, reaching 15–25 pmol/mL for free sterols in plasma. The optimized method is applied to the analysis of human plasma, leading to the identification of 92 lipid species in the targeted lipid classes. This represents a substantial improvement in sensitivity and detection capabilities compared to those of previously reported methods.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"183 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Digital Redepleted of Stimulated Emission Depletion Microscopy for Noise Reduction and Resolution Improvement","authors":"Xinwei Gao, Yong Guo, Luwei Wang, Yue Chen, Xiangcong Xu, Lukui Xu, Xiaoyu Weng, Wei Yan, Junle Qu","doi":"10.1021/acs.analchem.5c00101","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00101","url":null,"abstract":"Stimulated emission depletion microscopy (STED) achieves resolution beyond the diffraction limit by employing a donut-shaped depletion laser that selectively reduces fluorescence at the periphery of the excitation area. The imaging quality of STED microscopy is closely tied to minimizing the intermediate light from the ring-depletion laser. In this study, we introduce a method termed “digital redepleted STED,” which uses frequency domain filtering to generate an optimal donut profile by subtracting the “perfect donut” signal from the original STED data. This approach effectively reduces background noise and enhances the STED resolution. Through simulation experiments, we demonstrate that digitally redepleted STED doubled the resolution. This method is compatible with a wide range of biological samples and can be adapted for two-organelle-structure STED and 3D STED applications. We compare the performance of digitally redepleted STED with that of digitally enhanced STED (De STED) and deconvolution methods (STED Decon) in terms of the signal-to-background ratio (SBR) and resolution as evaluation metrics, and we find that our method doubled the resolution and SBR for different samples compared with origin STED. Our results indicate that digitally redepleted STED outperforms both De STED and STED Decon for complicated sample like mitochondria. We anticipate that the digitally redepleted STED will have broad applicability due to its enhanced resolution, improved SBR, and ease of implementation.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"26 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zhihao Zhang, Huaiwen Cang, Qingyun Li, Yi Yu, Huimin Jiao, Yuanyuan Xie, Shan Deng, Jichun Jiang, Lei Hua, Haiyang Li, Hang Li
{"title":"Miniature Thermoelectric Cooler for Long-Term Stable Dehydration for On-Site Breath Analysis by Direct Inlet Photoionization Ion Mobility Spectrometry and Mass Spectrometry","authors":"Zhihao Zhang, Huaiwen Cang, Qingyun Li, Yi Yu, Huimin Jiao, Yuanyuan Xie, Shan Deng, Jichun Jiang, Lei Hua, Haiyang Li, Hang Li","doi":"10.1021/acs.analchem.5c00138","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00138","url":null,"abstract":"Rapid and highly frequent measurements of exhaled breath hold significant value for disease diagnosis and exposure assessments. Direct inlet mass spectrometry and ion mobility spectrometry demonstrate great potential for on-site breath analysis, but their accuracy and sensitivity are notably affected by the high humidity in the breath samples. A miniature and continuous dehydration device based on dual-switching thermoelectric cold traps has been developed to efficiently reduce the humidity of breath gas from saturation to relative humidity of 4.3%, and a subcooling compensation method was adopted to eliminate the humidity fluctuations when switching the two cold traps to remove the condensed water in the trap, which could achieve a stable outlet humidity with RSD of 8.2% regardless of inlet humidity variations. The performance and profits of the miniature dehydration device for on-site analysis of breath samples have been tested by coupling with direct inlet photoionization ion mobility spectrometry and time-of-flight mass spectrometry. Trace breath acetone could be directly measured, and its concentration profiles over an 8 h period in fasting volunteers could be continuously monitored every 30 s. Furthermore, the <i>m</i>/<i>z</i> peaks from chlorinated hydrocarbons in exhaled breath samples were clearly identified after passing the dehydration device, which can be used for an occupational health assessment of the widely used halocarbon solvents.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"59 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"A New Chemiluminescence-Based Rapid Diagnostic Testing Platform with Sequential Dual-Flow Strips for Cardiac Troponin I (cTnI)","authors":"Heeyeong Jang, Supreeth Setty, Chong Ahn","doi":"10.1021/acs.analchem.4c06427","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06427","url":null,"abstract":"Although the most commonly used method for enhancing a limit of detection (LoD) in immunoassay is adopting chemiluminescence (CL), the liquid form of CL substrates has hindered its use for rapid diagnostic testing (RDT). In order to use the CL-based immunoassay in RDT with minimal user intervention, the liquid CL substrate should be converted to a dry form. In addition, a new RDT platform that is able to perform two sequential flows needs to be developed for the sequential flow control of the CL substrate. In this work, we have successfully developed a new dry form of CL substrate on the strip using a lyophilization process, as well as new lateral flow strips using an additional membrane pad for a time delay to achieve the desired sequential dual flows. Thus, on the dual-flow RDT strips, first the detection antibody conjugated with an enzyme flows over the test and control lines, and then the reconstituted CL substrate flows later. A hydrophilic PVDF membrane was selected as a pad material for the time delay to achieve the sequential dual flows through two flow paths, and flow introduction timing was functionally controlled to secure the time delay of approximately 5 minutes desired between the two flows. A CL-based cardiac troponin I (<i>cTnI</i>) assay was successfully performed on the new dual-flow RDT platform with a sample volume of 120 μL, achieving a LoD of 100 pg/mL. The achieved LoD is better than those possible with most of the currently available RDTs on the market. The new CL-based RDT platform with the capability of dual flows developed in this work can be used for numerous other immunodiagnostic platforms which need further high-sensitivity detection, envisaging a new RDT platform for point-of-care testing with further quantitative analysis.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"49 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723849","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Direct Detection of Natural-Abundance Low-γ Nuclei NMR Signals of Minute Quantities of Organic Solids","authors":"Zhiwei Yan, Jiangying Li, Hailu Zhang, Rongchun Zhang","doi":"10.1021/acs.analchem.4c06887","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06887","url":null,"abstract":"Low-γ nuclei signal enhancement in solid-state NMR spectroscopy is typically achieved via cross-polarization (CP) using abundant <sup>1</sup>H polarization in organic solids. Nevertheless, direct low-γ nuclei signal detection via a single CP process is quite challenging with minute quantities of samples due to the extremely limited signal-to-noise ratio (SNR) of the acquired spectra. Herein, we demonstrated the robust performance of a multiple-contact CP experiment with multiple acquisition periods (MCP) in each transient scan, leading to several-fold SNR enhancement over a conventional single-CP experiment at fast MAS conditions with slightly increased experimental time. Spin thermodynamic analysis was further performed to achieve maximum SNR by adding the obtained <i>N</i><sub>max</sub> CP spectra from each transient, where <i>N</i><sub>max</sub> ∼ <i>T</i><sub>1ρ</sub>/<i>τ</i><sub>cw</sub>. Here, <i>T</i><sub>1ρ</sub> is the proton spin–lattice relaxation time in the rotating frame, and <i>τ</i><sub>cw</sub> is the total time of CP and a heteronuclear decoupling period. The theoretical analysis is in good agreement with experimental results, and more than 4.5-fold SNR enhancement can be achieved for the pharmaceutical danazol/vanillin cocrystals. Besides, MCP was also used for proton <i>T</i><sub>1</sub> and <i>T</i><sub>1ρ</sub> measurement with high-resolution <sup>13</sup>C detection, where both proton <i>T</i><sub>1</sub> and <i>T</i><sub>1ρ</sub> can serve as the spectral-editing basis to identify different immiscible components in complex molecular systems.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"10 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Qing Dong, Xu Sun, Yuling Wang, Wei Zhang, Fan Feng, Dan Li, Jin Wang, Erkang Wang
{"title":"Enabling Sensitive Quantification of Exosomes Combining Aptamer-Based Rolling Circle Amplification and Silver Nanoparticles","authors":"Qing Dong, Xu Sun, Yuling Wang, Wei Zhang, Fan Feng, Dan Li, Jin Wang, Erkang Wang","doi":"10.1021/acs.analchem.4c06656","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06656","url":null,"abstract":"Exosomes carry various biological information and are abundant in body fluids, making them a promising noninvasive biomarker for disease diagnosis and prognosis. However, current detection methods have limitations in sensitivity, specificity, and cost effectiveness, hindering their clinical application. To address these challenges, we have developed a fast, accurate, and cost-effective method for detecting exosomes with high sensitivity and specificity, making it ideal for clinical applications. Clusters of differentiation 63 (CD63) aptamer with its complementary DNA (CD63 aptamer/cDNA) linked to streptavidin-coated magnetic beads (SA-MBs) are used as a capture probe. Exosomes with CD63 proteins can bind to the aptamer and release the cDNA, which initiates rolling circle amplification (RCA) to magnify the cDNA copies. The negatively charged RCA products induce the aggregation of positively charged spermine-modified silver nanoparticles (AgNPs) through electrostatic attraction. The aggregation of AgNPs can be observed visually with the naked eye or quantitatively analyzed using ultraviolet–visible (UV–vis) spectroscopy to determine the concentration of exosomes, with limits of detection of 4.0 × 10<sup>4</sup> particles/mL for visual observation and 800 particles/mL for UV–vis spectroscopy, respectively. The method has also been demonstrated for detecting the exosomes in serum samples, indicating its potential for clinical use in liquid biopsy.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"99 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nir Galili, Anna Somlyay, Giorgia Aquila, Reto Wijker, Philip Gautschi, Lukas Wacker, Jordon D. Hemingway
{"title":"High-Precision Oxygen-Isotope Analysis of Iron (Oxyhydr)oxides Using High-Temperature Conversion Isotope Ratio Mass Spectrometry","authors":"Nir Galili, Anna Somlyay, Giorgia Aquila, Reto Wijker, Philip Gautschi, Lukas Wacker, Jordon D. Hemingway","doi":"10.1021/acs.analchem.4c04676","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04676","url":null,"abstract":"We introduce a novel high-precision method for oxygen-isotope analysis of iron (oxyhydr)oxides using high-temperature conversion isotope ratio mass spectrometry (HTC-IRMS). In this approach, a finely ground mixture of iron (oxyhydr)oxide and graphite is heated at 1450 °C in a helium flow environment, converting oxygen to CO gas with nearly 100% yield. Continuous-flow IRMS analysis of the liberated CO yields a precision of ±0.15‰ (1σ, <i>n</i> = 28) and shows excellent agreement with (and improved precision over) traditional fluorination methods. This practical and safe technique expands access to oxygen-isotope measurements of iron oxides, thereby enhancing their utility in Earth and environmental sciences.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"7 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143723848","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jade K. Macdonald, Stephen C. Zambrzycki, Harrison B. Taylor, Jaclyn B. Dunne, Montana Quick, Anand S. Mehta, Richard R. Drake, Peggi M. Angel
{"title":"Optimization of Collagenase Proteomics for Improved Mass Spectrometry Imaging Peptide Identification","authors":"Jade K. Macdonald, Stephen C. Zambrzycki, Harrison B. Taylor, Jaclyn B. Dunne, Montana Quick, Anand S. Mehta, Richard R. Drake, Peggi M. Angel","doi":"10.1021/acs.analchem.4c04818","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04818","url":null,"abstract":"The extracellular matrix (ECM) is composed of a dynamically regulated collagenous scaffold that provides structure, conveys cellular and environmental communication, and contributes to disease progression. Collagen proteins derived from clinically archived formalin-fixed, paraffin-embedded (FFPE) tissues are analytically challenging due to dense post-translational modifications, high proline content, and insolubility. A recent advancement in ECM proteomics is the use of collagenase type III, an ECM-specific bacterial protease, to target native collagenous structures on-tissue for peptide imaging. The resulting collagenase-generated peptides have biochemical differences compared to tryptic peptides, creating analytical challenges in elucidating peptide sequence information. In this study, we characterize collagenase as a proteomic enzyme for ECM-targeted liquid chromatography trapped ion mobility spectrometry tandem mass spectrometry (LC-TIMS-MS/MS) and matrix-assisted laser/desorption ionization mass spectrometry imaging (MALDI-MSI) proteomic workflows. We then optimized collagenase-generated peptide sequencing for MALDI-MSI peptide identification from clinically archived FFPE tissue sections. Soluble rat tail collagen solution is used as a collagen standard to elucidate tryptic and collagenase cleavage sites within collagen. Proteomic readouts of FFPE tissue are compared across trypsin and collagenase digests to assess for ECM enrichment by collagenase in biologically complex samples. Optimized methods for MALDI-MSI peptide identification are comprehensively detailed from sample preparation to MS data acquisition and MS data analysis for reproducible implementation. On-tissue digestion followed by liquid surface extraction (LSE), inclusion of singly charged peptides during data acquisition, and implementation of nonspecific cleavage during database searching resulted in the most collagenase-generated peptide spectrum matches as well as MALDI-MSI peptide identifications. This research establishes parameters for the optimal identification of peptides from collagenase-directed ECM proteomic workflows for targeted spatial analysis of the ECM.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"36 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}