Mass Spectrometric Analysis of Protein-Nucleic Acid Cross-links . 21

of polymers, exhibiting different physicochemical properties therefore they require different conditions for ionization in MS. The peptides and oligonucleotides are ionized in positive and negative ion modes respectively. During the current studies the interest lies in the identification of the protein region interacting with RNA therefore the ionization is performed in positive ion mode (Schmidt et al.,

22 2012). However, the presence of excess of non-cross-linked components hampers the ionization of the cross-links. The increased hydrophilicity of peptide-oligonucleotide heteroconjugates due to presence of peptide-oligonucleotide moiety cross-linked to the peptide in comparison to the unmodified non-cross-linked peptides may cause lower ionization efficiency. For the relative ionization improvement, the oligonucleotide part of the cross-linked heteroconjugate should be made as small as possible, maximum up to four nucleotides by using nucleases (Steen & Jensen, 2002; Qamar et al., 2015).

For the mass spectrometric analysis of peptide-oligonucleotide heteroconjugates, the HCD fragmentation has proved to be better than the CID fragmentation methods. The mass spectrometers with orbitrap analyzers carry out the HCD fragmentation with high accuracy. It helps in differentiating the distinct signals generated by peptide and nucleotide fragmentation such as the signals of immonium ion of tyrosine (m/z 136.0762) and the RNA marker ion of adenine (m/z 136.0623). In addition, the peptide-oligonucleotide heteroconjugate spectrum generated by HCD fragmentation usually has long y-ion series, high intensity signals of a2 and b2 ions, signals of immonium and internal ions and nucleic acid marker ions that improve its identification.

1.5.1.4 Protein-Nucleic Acid Cross-links Data Analysis

The low signal intensity in MS/MS spectrum and the wide variety of potentially cross-linked nucleotide fragments has made the interpretation of the data obtained by the mass spectrometric analysis of protein-nucleic acid cross-linking, very challenging and laborious. The cross-linking is usually an additive reaction.

The molecular weight of the peptide-oligonucleotide heteroconjugate is the sum of the molecular weight of the peptide and the oligonucleotide moiety cross-linked to it. The MS/MS spectra obtained are usually prevailed by the signals of peptide fragments. In this case, the cross-linked nucleotide moiety can only be deduced by calculating the mass difference between the experimental peptide-oligonucleotide heteroconjugate and the peptide (Kramer et al., 2011).

23 In recent years, a semi-automated data analysis approach has been developed for the unbiased analysis of peptide-oligonucleotide heteroconjugates. It is comprised of RNP^xl tool (Kramer et al., 2014) in OpenMS environment (Sturm et al., 2008; Bertsch et al., 2011) using OMSSA (Geer et al., 2004) as the search engine. The oligonucleotide fragment mass is dealt as variable modification while searching for PTM against database. After endonuclease digestion and TiO2

enrichment, the heteroconjugates with maximum four nucleotides are possible.

Taking this into consideration, 69 different mass combinations out of four different nucleotides and 829 different mass combinations out of four different nucleotides along with RNA/DNA modifications like loss of H2O and HPO3 etc. are possible.

During the database search, these mass variants are used to generate the theoretical precursor fragment spectra for every original spectrum. By subtracting the molecular weight of the oligonucleotide from the experimentally determined molecular weight of heteroconjugate, the molecular weight of the peptide can be deduced. The sum of combination of oligonucleotide fragment mass along with peptide mass and spectrum which fits to the experimental precursor mass along with its candidate spectrum will yield a most probable hit. Additionally, different filters can be applied according to the experimental design such as for comparing the experimental sample with the control one and also for removing the pure peptide hits etc. (Kramer et al., 2014). The manual inspection of the MS/MS spectrum is immensely important to screen the exact amino acid and nucleotide, undergone cross-linking reaction. The presence of signals of marker ions of nucleic acid base resulting by the nucleic acid fragmentation and the shift of b or y ion series or signals of immonium and internal ions by the mass of cross-linked nucleotide fragment or adduct, indicate the cross-linked amino acid along with cross-linked nucleotide (Qamar et al., 2015). Oftenly, the cross-linking bond formed between peptide and oligonucleotide is labile to HCD fragmentation resulting in the identification of the cross-linked peptide but the identification of the single cross-linked amino acid residue is no longer possible. So far, the unavailability of a software that can handle all the aspects of peptide-oligonucleotide heteroconjugate fragment spectra makes the requirement of the

24 completely automated system for protein-nucleic acid cross-linking data indispensable.

Figure 1.4: Workflow of cross-linking protocol (Figure adapted from Qamar et al., 2015). The in vitro transcribed MS2-tagged pre-mRNA is incubated with HeLa nuclear extract for the RNP complex assembly. The assembled RNP complex is purified. The purified RNP complex is then UV-cross-linked. The proteins in the sample are digested with trypsin. The cross-links are isolated and the non-cross-linked peptides are removed by administering the sample to the SEC. The RNA is hydrolyzed by the RNases. The non-cross-linked RNA oligonucleotides are removed by RPC. The RNA-protein cross-links are subsequently enriched by using TiO2 solid phase extraction. The sample is then subjected to LC-ESI-MS/MS analysis. The RNP^xl pipeline in an OpenMS environment is used to analyze MS data.

25

1.6 Biological Complexes Studied Using UV-Induced