Limbach Group Biological Mass Spectrometry

Cellular processes, such as replication, packaging, repair and transcription of DNA, and maturation, transport, and translation of RNA, require extensive interaction of nucleic acids with various proteins. A fundamental prerequisite for understanding the interactions between proteins and nucleic acids is a detailed knowledge of the structure of nucleic acid-protein complexes. Ribosomes are RNP complexes composed of rRNAs and proteins. The ribosome holds the central role of linking the worlds of nucleic acids (DNA and RNA) with proteins. It serves as the scaffold to translate information contained in messenger RNA (mRNA) into the corresponding sequence of amino acids. The ribosome carries out the task of translation with amazing accuracy and speed: 10-20 amino acids are incorporated per second into the nascent polypeptide chain with only one error in every 3000 codons deciphered. To help understanding of how ribosomes are formed and the important RNA-protein interactions that must occur to generate functional complexes, our research program in this area uses a variety of E. coli mutants to capture transient complexes that are involved in the pathway of ribosome formation. To characterize the RNA-protein interactions, we have developed - in collaboration with Prof. Joe Caruso at UC - a combined ICP and LC-ESI-MS approach for RNA:protein cross-link detection and identification. 

Relevant Publications

• Sharma, I.M.; Rappé, M.C.; Addepalli, B.; Grabow, W.W.; Abeysirigunawardena, S.C.; Limbach, P.A.; Jaeger, L.; Woodson, S.A.; “A metastable rRNA junction essential for bacterial 30S biogenesis” Nucleic Acids Research (2018) 46 5182-5194.PMID: 29850893  
• Dator, R.P.; Gaston, K.W.; Limbach, P.A.; “Multiple Enzymatic Digestions and Ion Mobility Separation Improve Quantification of Bacterial Ribosomal Proteins by Data Independent Acquisition Liquid Chromatography−Mass Spectrometry” Analytical Chemistry 86 (2014) 4264-4270. PMID: 24738621.      
• Wong, S.Y.; Javid, B.; Addepalli, B.; Piszczek, G.; Strader, M.B.; Limbach, P.A.; Barry III, C.E. “Functional Role of Methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis” Antimicrobial Agents and Chemotherapy 57 (2013) 6311-6318. PMID: 24100503.   
• Clatterbuck Soper, S.F.; Dator, R.P.; Limbach, P.A.; Woodson, S.A.; “In vivo X-ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates”, Molecular Cell 52 (2013) 506-516. PMID: 24207057.    
• Catron, B.L.; Caruso, J.A.; Limbach, P.A.; “Selective Detection of Peptide-Oligonucleotide Heteroconjugates Utilizing Capillary HPLC-ICPMS”, Journal of the American Society for Mass Spectrometry. 23 (2012) 1053-1061. PMID: 22451333. 
• Easter, R.N.; Kröning, K.K; Caruso, J. A.; Limbach, P.A.; “Separation and identification of oligonucleotides by hydrophilic interaction liquid chromatography (HILIC) - inductively coupled plasma mass spectrometry (ICPMS)”, Analyst 135 (2010) 2560-2565. PMID: 20830328.  
• Krivos, K., Limbach, P.A. “Sequence Analysis of Peptide:Oligonucleotide Heteroconjugates by Electron Capture Dissociation and Electron Transfer Dissociation”, Journal of the American Society for Mass Spectrometry 21 (2010) 1387-97. PMID: 204335485.    
• Brock, J.E.; Pourshahian, S.; Limbach, P.A.; Janssen, G.R. “Ribosome Binding to Leaderless mRNA through Recognition of their 5’-terminal AUG”, RNA 14 (2008) 2159-2169. PMID: 18755843.