The mass spectrometric characterization of protein:RNA cross-links is challenging due to the heterogeneity of the sample and the physical and chemical properties of the cross-link components. This dissertation aims at overcoming these challenges by developing an analytical approach for the selective detection and quantification of peptide-oligonucleotide heteroconjugates and protein:RNA cross-links. The methods developed were adapted from previous studies and take advantage of the dual nature of the cross-link, where one set of chromatographic conditions separate based on the peptide portion of the cross-link, while a complementary set of chromatographic conditions is used based on the oligonucleotide portion of the cross-link. These liquid chromatography (LC) conditions were optimized first using synthetic oligonucleotide:peptide heteroconjugates, and then scaled from capillary to conventional conditions to enable split flow and fraction collection. Scaling up the chromatography and applying a 1:100 split allows for detection of the phosphorus response via the ICPMS, while the majority of the fraction is collected for subsequent liquid chromatography with tandem molecular mass spectrum analysis.
Inductively coupled plasma mass spectrometry (ICPMS) is a powerful analytical tool for element specific analysis. Instrumental advances, such as the collision/reaction cell (CRC), gave rise to monitoring a variety of previously unattainable elements, which include sulfur and phosphorus, while increasing the sensitivity of overall metal detection. Furthermore, ICPMS is capable of element-specific detection, where the intensity of the element is directly proportional to the element present in the sample allowing for quantification of elements of interest. In addition, ICPMS ionization is neither matrix- nor amino acid sequence-dependent, thus removing ionization issues most commonly seen in traditional mass spectrometry approaches.
The methods were then applied to oligonucleotide:peptide heteroconjugates generated from cross-linking between messenger RNA (mRNA) and ribosomal protein S1 formed through 4-thiouridine as the cross-linking reagent, as they contain phosphorus (from the oligonucleotide) and sulfur (from the cross-linking reagent). The selective purification of ribosomal protein S1:leaderless mRNA cross-links using ICPMS allows for the purified fraction to be further analyzed by LC-MS/MS for sequence analysis. Once sufficient sample was collected, the cross-links were digested using nuclease P1 (to reduce the size of the mRNA mimic to a monomer) and the cross-link is then sequenced using collision induced disassociation to identify the amino acid residues in ribosomal protein S1 involved in cross-linking. This novel method will enable identification of the binding sites on S1 and provide pertinent information on how this ribosomal protein recognizes mRNAs lacking the Shine-Dalgarno sequence.