Abstract This grant application requests funds to purchase a Thermo Scientific Orbitrap Exploris 480 and UltiMate 3000 RSLCnano HPLC, and FAIMS (high field asymmetric ion mobility spectrometry) interface for shared use in the Institutional Mass Spectrometry Laboratory (MSL) at the University of Texas Health Science Center at San Antonio (UTHSCSA), directed by Dr. Susan Weintraub. The requested system will provide enhanced capabilities and expanded capacity for protein identification and relative quantification as well as valuable information for structure determination of proteins and protein complexes. Project summaries for 14 investigators are presented, with major focus on cancer and infectious disease (HIV, TB and SARS-CoV-2). The global proteomics experiments proposed by the user group will be conducted by data-independent acquisition mass spectrometry (DIA-MS). Use of this technique in the MSL over the past three years has demonstrated its extraordinary capabilities for comprehensive protein identification with greater accuracy for relative quantification, in an efficient and remarkedly reproducible manner. DIA-MS is being used in the MSL for numerous studies requiring discovery proteomics [including analysis of proteins from cells, tissues, secretomes, alveolar lining fluid (ALF)] and for interactomics. Cross-linking-MS is a new service that will be offered by the MSL; it will be an important partner for protein structure analyses conducted by cryo-electron microscopy in the new UTHSCSA facility that will open in spring 2022. In 2020, the user group of the MSL submitting samples for protein/peptide analysis consisted of 27 individual investigators in 17 departments at UTHSCSA in addition to 11 researchers from nine different outside institutions. Submissions to the MSL for protein identification/relative quantification dramatically increased when investigators became aware of the incredible power of DIA-MS. This resulted in a progressively growing backlog of DIA-MS samples. The level of interest in DIA-MS is evidenced by the support letters written for grant applications by Dr. Weintraub: >70% of the 35 letters written in 2020 discussed DIA-MS experiments (in most cases as the only MS-based technique being planned). DIA-MS analyses are currently being conducted on a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer, yielding previously unprecedented numbers (>5,000) of protein identifications and relative quantifications for single injections of cell lysate samples. Results acquired in the Thermo “demo” lab showed that the requested Exploris outperformed the Lumos for DIA-MS analysis of aliquots of the same samples (6,665 vs 5,248 proteins). Acquisition of the requested Exploris is essential to handle the rapidly-expanding submissions and increasing backlog for DIA-MS in the MSL as well as to provide new capabilities for protein/protein complex structure analysis via cross-linking-MS. The analytical support that will be provided by...