Identification of Trna Modifications in T. Thermophilus: Wild Type HB8 and Mutant DTTHA1897 by LC-UV-MS/MS

Identification of tRNA modifications in T. thermophilus: wild type HB8 and mutant DTTHA1897 by LC-UV-MS/MS

A Thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of

Master of Science In the Department of Chemistry Of the College of Arts and Sciences by

Lihua Fu

Bachelor of Science from University of Science and Technology Beijing, China 2008

Abstract

Transfer RNAs (tRNAs) are essential adapter molecules for translation of the genetic code into a peptide sequence at the ribosome. The study of tRNA modifications are essential subjects of bioscience and are waiting to be explored in the areas of identification, function, structure, biosynthesis and the sequence of tRNA, and the locations of the post-transcriptional modification on tRNA sequences. Thermus thermophilus wild type and ΔTTHA 1897 were the model system that used to peruse a better understanding on tRNA modifications in this work. TTHA1897 is a putative gidA gene, which is one of the two crucial biosynthesis enzyme genes for xm5s2U in E. coli.

ΔTTHA 1897 is mutant strain with disruption of tRNA gene TTHA1897 in T. thermophilus.

Growth phenotype studies including growth rate and temperature sensitivity studies demonstrated that ΔTTHA1897 experienced a slower growth rate and non-survival in minimal medium. An HPLC-UV-MS study identified mnm5s2U in the wild type but not in

ΔTTHA1897. Taking advantages of FT-ICRMS, the unique fragmentation pattern of xm5s2U was discovered with a proposed mechanism. An effective SRM method was developed and tested using E. coli tRNA nucleosides based on the unique fragmentation pattern of xm5s2U. A triple quadrupole mass spectrometer was also used to investigate a neutral loss method for the identification of xm5s2U nucleosides.

List of Abbreviations RNA Ribonucleic Acid

DNA Deoxyribonucleic Acid mRNA Messenger RNA tRNA Transfer RNA rRNA Ribosomal RNA

T. thermophilus Thermus thermophilus

E. coli Escherichia coli

TEM Thermus Enhanced Media

LC-MS Liquid Chromatography coupled with Mass Spectrometry

ESI Electrospray ionization

CID Collision induced dissociation

MS/MS Tandem mass spectrometry

SRM Select reaction monitoring

MRM Multiple reaction monitoring

FT-ICR Fourier transform ion cyclotron resonance

A Adenine

C Cytosine

G Guanine

U Uracil

I Inosine cmnm5U 5-Carboxymethylaminomethyluridine

2 mnm5U 5-Methylaminomethyluridine nm5U 5-Aminomethyluridine cmnm5s2U 5- Carboxymethylaminomethyl-2-thiouridine mnm5s2U 5-Methylaminomethyl-2-thiouridine nm5s2U 5-Aminomethyl-2-thiouridine

D dihydrouridine

Ψ pseudouridine

Q queuosine cmo5U uridine 5-oxyacetic acid acp3U 3-(3-amino-3-carboxypropyl)uridine mcm5U 5-methoxycarbonylmethyl-uridine m1G 1-methylguanosine m2G 2-methylguanosine m7G 7-methylguanosine

Gm 2'-O-methylguanosine ac4C N4-acetylcytidine k2C lysidine

Cm 2'-O-methylcytidine m5U 5-methyluridine

Um 2'-O-methyluridine

S2U 2-thiocytidine s4U 4-thiocytidine

3 m5s2U 5-methyl-2-thiouridine t6A N6-threonylcarbamoyladenosine ms2t6A 2-methylthio-N6-threonyl carbamoyladenosine m2A 2-methyladenosine m1A 1-methyladenosine m6A N6-methyladenosine io6A N6-(cis-hydroxyisopentenyl)adenosine

6 6 6 m 2A N , N –dimethyladenosine ms2i6A 2-methylthio-N6-isopentenyladenosine ms2io6A 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine i6A N6-isopentenyladenosine

Acknowledgement

First of all, I would like to thank my advisor Dr. Limbach! He is one of the most supportive and smart teacher ever. I would never forget the moments of his kind, patience, and encouraging words when I was frustrated with my progress. Any time, he is always there to support and encourage. Especially, he guided me through the down times. I express my appreciation to him for his mentoring strategy. He always leaves so much room for me to discover through experimenting, but he is always there when I need help.

I would like to send my heartfelt thanks to my committee members Dr. Heineman and

De. Merino. Dr. Heineman’s kind words and guidance helps me very much on some daunting research subjects. Dr. Merino always helps me think about more aspects of my research. Without their long-term commitment of helping, I would never be able to go this far.

A special thanks to Dr. Larry Sallans for his kindness and expertise while helping me with the instruments. I would like to thank the Limbach group members, Dr. Balu Addepalli,

Dr. Kirk Gaston, Dr. Stephen Macha, Dr. Siwei Li, Dr. Romel Dator, Dr. Rebecca Rohlfs,

Dr. Susan Russell, collin Wetzel, Wunan Shi, Xiaoyu Cao, Rob Ross, Yang Jiao. Dr. Gaston provided very valuable comments on many areas of my researches.

I would also like to give special thanks to my friends, Dr. Tingting Wang, Dr. Li Duan,

Wunan Shi, and Xiaoyu Cao for their continuous support during my time in University of

Cincinnati.

I greatly appreciated continuous care and support from my family. My dearest parents and parents in law took excellent care of my children while I was working on test, research and teaching. My husband Dr. Xiaoping Chen is always there lending me a shoulder to rest. My two little girls are the most inspiring kids to mommy ever.

Table of Contents Abstract ...... 1 List of Abbreviations ...... 2 Acknowdgement ...... 5 Chapter 1. The Significance of tRNA ...... 9 1.1 RNA Structure and Type...... 9 1.2 Transfer RNA Structure and Function ...... 10 1.3 tRNA Modifications and Their Significance ...... 10 1.4 Methods in Studying tRNA Modifications ...... 21 Chapter 2 Phenotype of T. Thermophilus HB8 Wild Type and ΔTTHA 1897 ...... 23 2.1 Introduction ...... 23 2.2 Materials and Methods ...... 23 2.2.1 T. thermophilus HB8 Wild Type ...... 24 2.2.2 The Construction of ΔTTHA1897 Disruption Mutant ...... 24 2.2.3 Total tRNA Isolation ...... 25 2.2.4 Growth Conditions ...... 26 2.3 Results and Discussion ...... 26 2.3.1 Total tRNA Isolation ...... 26 2.3.2 Phenotypes ...... 27 2.3.3 Discussion ...... 29 2.4 Conclusions ...... 30 Chapter 3 Identification of tRNA Modifications in T. thermophilus and Discovery of the Fragmentation Pattern of xm5s2U Nucleosides Family ...... 31 3.1 Introduction ...... 31 3.2 Materials and Methods ...... 31 3.2.1 Enzymatic Digestion ...... 31 _Toc425154930 3.2.2 HPLC-UV-LC/MS Analysis of Nucleosides ...... 32 3.2.3 Mass Condition ...... 34 3.3 Results and Discussion ...... 34 3.3.1 HPLC-UV-LCMS Analysis of Nucleosides ...... 34 3.3.2. Discussion ...... 37

3.4 Conclusions ...... 38 Chapter 4. The Fragmentation Pattern of xm5s2U Nucleosides ...... 39 4.1 Introduction ...... 39 4.2 Materials and Methods ...... 39 4.2.1 Materials ...... 39 4.2.2 HPLC-UV-LCMS Analysis of xm5s2U Standards ...... 39 4.2.3 Direct Infusion Electrospray Mass Spectrometry ...... 39 4.3 Results and Discussion ...... 40 4.3.1 LC-MS Analysis of xm5s2U Standards ...... 40 4.3.2 Direct Infusion of xm5s2U Standard on FT-ICRMS ...... 41 4.4 Conclusions ...... 50 Chapter 5 SRM method for the study of xm5s2U family nucleosides ...... 51 5.1 Introduction ...... 51 5.2 HPLC-UV-MS Instrumental Conditions ...... 51 5.3 Result and Discussion ...... 52 5.4 Conclusion ...... 54 Chapter 6 Neutral loss on Triple quadrupole mass spectrometer ...... 55 6.1 Introduction ...... 55 6.2 Experimental ...... 55 6.2.1 Materials and Instrument Conditions ...... 55 6.2.2 MRM and Neutral loss Scan Set Up ...... 56 6.3 Results and discussion ...... 58 6.3.1 Experiment 1: MS Scan, MRM and Neutral Loss Scans ...... 58 6.3.2 Experiment 2: MRM and Neutral loss scans without MS scan ...... 65 6.4 Conclusions ...... 70 Chapter 7 Conclusions and Future Work ...... 71 7.1 Thesis Conclusions ...... 71 7.2 Future work ...... 72 References ...... 74 Appendix I ...... 77 Appendix II ...... 81

Chapter 1. The Significance of tRNA

1.1 RNA Structure and Type. As one of the three major bio-macromolecules (along with deoxyribonucleic acid and proteins), ribonucleic acid (RNA) is made up of a long chain of components called nucleotides. Each nucleotide consists of a nucleobase, a phosphate group, and a ribose sugar moiety of which carbons are numbered 1' through 5' (Figure 1.1). A base is attached to the 1' position, and a phosphate group is attached to the 3' position of one ribose and the 5' position of the next. In general, the bases of RNA are adenine (A), cytosine (C), guanine (G), and uracil (U), but these bases and attached sugars can be modified in many ways resulting in a variety of nucleotide structures. The modifications are often derived from the four normal nucleosides, adenosine, guanosine, cytidine and uridine(1, 2).

7 6 N 5 NH 1 8 O 9 4 2 N HO P O 5' N NH2 O 3 O 4' H H 1' Base Phosphate H 3' 2' H (Guanine) OH OH

Sugar

Figure 1.1. Structure of a nucleotide

There are three major types of RNA molecules: messenger RNA (mRNA), transfer RNA

(tRNA) and ribosomal RNA (rRNA). mRNA molecules are the information-carrying

9 intermediates in protein synthesis, which direct the assembly of proteins on ribosomes. tRNA molecules deliver amino acids to the ribosome for peptide bond formation in a sequence dictated by the mRNA template. rRNA is the major component of ribosomes, which plays both a catalytic and structural role in linking amino acids together to form proteins.

1.2 Transfer RNA Structure and Function Transfer RNAs (tRNAs) are essential adapter molecules needed for the translation of the genetic code into a peptide sequence at the ribosome. To better understand how tRNA functions in protein synthesis, the Central Dogma of Molecular Biology (3) proposed by

Crick is presented in Figure 1.2. DNA is transcribed into mRNA which is then translated into proteins. RNA can be divided into two categories based on its functions, protein- coding RNA (messenger RNA) and non-coding RNA. Proteins are synthesized according to the sequence information in mRNA by the translational machinery. Noncoding RNAs, although not translated into proteins, play a vital role in regulating and facilitating translation (4, 5). As an important species of noncoding RNA, transfer RNA (tRNA) brings the amino acid specified by the codon on mRNA to the growing peptide chain.

Figure1.2. Central Dogma of Molecular Biology

A typical tRNA has four short base-paired stems and four loops. The amino acid is bound at the 3’ end of the acceptor stem. The anticodon is the three base region (position 34,

35, 36) that can base pair to the corresponding three base codon region on mRNA. The structure of yeast tRNAphe is shown in Figure 1.3(6).

Figure 1.3. Tertiary and Secondary structures of tRNAPhe from yeast

There are 64 codons in the codon box including 3 stop codons. Nevertheless, only 20 amino acids are involved in translation. As show in Figure 1.4, multiple codons can represent one amino acid. Figure 1.4 is the universal genetic code(7).

Figure 1.4. The universal genetic code.

Different tRNAs which can be charged with the same amino acid are called isoacceptor tRNAs. Isoacceptors help explain the translation process. The number of tRNA species is always fewer than 61 codons, meaning that some tRNAs must decode more than one codon. In 1966, Crick proposed his wobble hypothesis, stating that the third position of codons could wobble base pair with the first position of the anticodon, which is the 34 position of tRNA. Wobble base pairing does not follow Crick base pair rules(8) (as shown in Table 1.1).

Table 1.1. The pairing rules between the third base on the codon and first of anticodon

Base on the anticodon Bases recognized on the codon

U A, G

C G

A U

G U, C

I U, C, A

Revised wobble hypotheses were later proposed to explain particular base pairing of tRNA with modifications at the wobble position based on in vitro experiments(9), as shown in Table 1.2.

Table 1.2. Revised wobble rule

Revised wobble rule

Anticodon N34 Codon N3

G U, C

C G

A U, C, (A), G

Unmodified U U, (C), A, G

xm5s2U, xm5Um, Um, xm5U A, (G)

xo5U U, A, G

I U, C, A

k2C A

1.3 tRNA Modifications and Their Significance To date tRNAs are intensively modified post-transcriptionally and more than 95 modified nucleosides are found in tRNA (2, 6, 7, 10, 11). Figure 1.5 is a typical tRNA secondary structure with modifications found at specific locations (9).

Figure 1.5. tRNA secondary structure with modifications found at specific locations.

The position of the modification could be on the base, sugar or both sugar and base.

Figure 1.6 is an example of modified nucleosides. The modification could be located at the T loop, D loop, and on the anticodon loop of the tRNA.

2‘-O-methylcytidine 1-methyladenosine 1 (Cm) (m A) N2,2'-O-dimethylguanosine 2 (m Gm )

Figure 1.6. Example of the modified nucleosides

There has been a great deal of interest in the role of post-transcriptional modifications in the function of tRNA. Modifications are essential to tRNA folding, secondary and

15 tertiary structure, and thus tRNA function (7, 12-15). As tRNA serves the function of decoding the genome, the tRNA modifications have the following functions, such as enhancement of ribosome binding affinity, reduction of misreading and modulating frame-shifting, and all of above functions affect the rate and fidelity of translation (16-

18). The modifications of the anticodon stem and loop regulate gene expression via ordering confirmation and dynamics for recognition of codons.

Ile 2 For example, tRNA CAU has a modified k C at the anticodon position 34. The

Ile modification specifies tRNA CAU to decode AUA. On the contrary, without the modified k2C, CAU would base pair with AUG which is a methionine codon. Furthermore, the modification is able to maintain the reading properties. For instance, the presence of mnm5s2U34 counteracts +2 ribosomal frameshifting while the absence of mnm5s2U34 in E. coli tRNAs of lysine, glutamine, glutamic acid, arginine and leucine promotes +1 mRNA-programmed frameshifting as shown in Figure 1.7(19-21).

modification Codon reading Frameshifting

5 2 mnm s U34 NNA/G +1/+2 2 k C34 AUA Figure 1.7. Examples of the rules of modifications in decoding

A tRNA’s anticodon is substantially structured by post-transcriptional modifications at position 34 and position 37. Modifications at these positions pre-structure the anticodon domain to ensure the correct codon binding (12). However, only limited information is available as to how these modifications influence the translation of

16 genetic information, the folding of tRNA, and tRNA function. tRNA modifications also play a role in tRNA aminoacylation as positive or negative determinants. Compared with mnm5s2U34 in E. coli tRNALys, the unmodified U34 reduces lysine acceptance activity by

140-fold, whereas, m1G37 in yeast tRNAAsp could restrict the efficiency for tRNA to be charged with a non-cognate amino acid (22). tRNA modifications can also serve a role structurally and confirmationally. For example,

Cm, Gm, s2U, m5s2U could thermo-stabilize tRNA; s4U could help define L shape which is the three dimensional tRNA structure(23). tRNA modifications are related with diseases as well. In one of the human mitochondrial diseases MERRF (Myocolnus epilepsy associated with ragged-red fibers) mutation, the mitochondrial tRNALys was lacking

τm5s2U. Lack of the modification makes tRNALys fail to translate codons AAA and AAG

(24). Other than that, researchers found that tRNA modifications are also involved in apoptosis regulation through binding with cytochrome C (25). Because of the essential role of tRNA modifications, determination of the modifications present in a tRNA, their position in the sequence, the extent to which they are modified, and how they are modified is crucial to the study of post-transcriptional modification in tRNA.

My thesis is focused on the modified nucleoside family xm5U and xm5s2U, which includes 5-carboxymethylaminomethyluridine (cmnm5U), 5-methylaminomethyluridine

(mnm5U), 5-aminomethyluridine (nm5U), 5-carboxymethylaminomethyl-2-thiouridine

(cmnm5s2U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), and 5-aminomethyl-2- thiouridine (nm5s2U). The biosynthesis pathway of the xm5s2U family of nucleosides is

17 shown in Figure 1.8. After transcription uridine will first be enzymatically modified to cmnm5U. For the tRNAs specific for glutamine, lysine, glutamate, and arginine, the cmnm5 group is further modified to an mnm5 group, probably for the purpose of enhancement of the stabilization of the modified group and efficiency of translation (18).

5 In bacteria and mitochondria, as well as some archaea, the x of xm U34 can be an amino

(nm5U), methylamino (mnm5U), or carboxymethylamino (cmnm5U) group as shown in

Figure 1.8. The thiolation of the nucleosides could occur anytime during the synthesis.

The synthesis mechanism is complex and will be discussed later.

Figure 1.8 Biosynthesis pathway of the nucleosides

xm5s2U modifications of nucleosides in the anticodon loop are important for tRNA recognition by cognate aminoacyl-tRNA synthetases and for accurate mRNA decoding.

For instance, mnm5s2U could serve as a tRNA positive determinant：compared with the

5 2 Lys native mnm s U34 in E. coli tRNA , the unmodified U34 reduces lysine acceptance

18 activity by 140-fold (23). The mnm5s2U also plays an important role in maintaining the reading frame because it can counteract +2 ribosomal frameshifting (26). On the other hand, mRNA-programmed +1 frameshifting was promoted by the absence of the

5 2 5 2 modification mnm s U34 (27). The function of xm s U in decoding is strongly restricted on wobbling. The unmodified U34 can base pair with U, C, G, A nucleosides on the third position of codon on mRNA. The cmnm5U at tRNA wobble position 34 reads codons ending with A or G in two-codon sets and prevents frameshifting (17). However, for the tRNAs specific for glutamine, lysine, glutamate and arginine, the cmnm5 group is further modified to an mnm5 group to enhance the stabilization of the modified group and efficiency of translation (18). The 2-thiolation relieves steric interactions between the large S atom with the 2’-OH while 5-methylamionomethyl cooperates with the 5’- phosphate group; 5-aminomethyl (-NH+): forms hydrogen bond with the 2’-OH of U33 which can be seen in Figure 1.9.

Figure 1.9. Crystal structure of mnm5s2U

The enzymatic synthesis of cmnm5U in E. coli involves the enzymes MnmE and GidA, and donor cofactors tetrahydrofolate derivative and glycine, and taurine in mitochondrial tRNA of higher animals (18, 28-30). This cmnm5U modification is one of the rare examples in which an amino acid is incorporated into a nucleotide modification. The detailed mechanistic reactions by MmnE and GidA have yet to be characterized.

Scientists still do not know how many steps precede the formation of cmnm5U. There are two proposed reaction mechanisms, both of which are based on the results of null mutagenesis of enzymes. The first proposal is that MnmE activity proceeds through

GidA in the MnmE/GidA pathway. The hypothesis was originated from the differences in the growth rates between E. coli mnmE and gidA null mutants. In the wild type E. coli, tRNAArg was observed to have mnm5UCU in the anticodon. The frameshift frequency of tRNAArg in the double (null gidA and mnmE) mutant is the same as that in the single mnmE mutant, but significantly lower than the frequency in the single GidA mutant (26,

31). The other proposal is that MnmE and GidA form a functional complex in which both proteins are interdependent. The proposed mechanism is based upon observations that no differences were detected between the growth rates of mnmE and gidA mutants in minimal medium, and an experiment where no intermediate with 5-substituents was observed in the gidA null mutant (32).

In T. thermophilus HB8, two gidA like genes, TTHA1897 and TTHA 1972 were found by a

Blast search; one mnmE like protein gene is TTHA 0931. In this study, the gidA like gene

TTHA1897 is substituted by a kanamycin resistance gene. The identification of xm5s2U modifications in tRNA was conducted by mass spectrometry. Mass spectrometry is a major analytical tool for the identification of tRNA modifications due to low sample consumption and high sensitivity. In this thesis, tRNA was isolated from the bacterium T. thermophilus, digested to nucleosides, analyzed by either a Thermo LTQ or Waters

Micromass Quattro LC mass spectrometer. Standards of xm5s2U were analyzed to assist the identification of low abundance xm5s2U in the biological sample.

1.4 Methods in Studying tRNA Modifications

The study of tRNA modifications has always been an interesting topic among researchers. Several methods have been developed to characterize tRNA modifications.

Due to the low abundance of some modifications, high sensitivity methods are required.

For example, the post labeling technique was conducted according to the trialcohol procedure of Randerath (33) and 5’ post labeling of 3’ mononucleotides was achieved using [ϒ-32P] ATP and T4 polynucleotide kinase (34, 35). There are some limitations in these two methods. The trialcohol procedure is not applicable to all nucleosides, as some thiolated nucleosides could be destroyed by this method. With T4 polynucleotide kinase, some nucleosides could not be easily phosphorylated. High-resolution thin layer chromatography is a method for separating, identifying and quantifying modifications.

However, it requires large amounts of material and has in vivo and in vitro limitations

(36-41).

Liquid Chromatography coupled with Mass Spectrometry (LC-MS) as a modern analytical tool was widely used for identification and quantification of tRNA modifications by

21 taking advantage of the separation ability of the front LC system and mass measurement of mass spectrometer. In 1982, Martin and coworkers developed and optimized the method of digesting total tRNA to nucleosides which were then analyze by reverse phase liquid chromatography (42). Liquid chromatography coupled with electrospray ionization mass spectrometry is utilized in my thesis research.

In this thesis work, T. thermophilus wild type and ΔTTHA 1897 strains were used to study the xm5s2U nucleosides, the existence in both wild type and ΔTTHA 1897, and the possible function of xm5s2U. Growth phenotype studies including growth rate and a temperature sensitivity study. tRNA was isolated from both strains and digested to nucleosides for further HPLC-UV-MS study. A total of 28 tRNA nucleosides were identified in the wild type including mnm5s2U and inosine. However a total of 26 tRNA nucleosides were identified in ΔTTHA1897, which was missing mnm5s2U and inosine.

By using FT-ICR MS, the accurate mass of all fragments was measured providing the molecular formula of each fragment. The fragmentation pattern of xm5s2U was proposed and used to develop an effective SRM method. Later, a Triple Quadrupole mass spectrometer was used to create a neutral loss method for identification of xm5s2U nucleosides.

Chapter 2 Phenotype of T. Thermophilus HB8 Wild Type and ΔTTHA 1897

2.1 Introduction

To study tRNA modifications, T. Thermophilus is used as the model system in this study, and cells are cultured to harvest tRNA. To investigate the role of tRNA modifications in T. thermophilus, a mutant strain was constructed. The phenotypes are standard microbiological studies as a quick, easy and intuitive way to investigate the gene function of the bacteria. In this study, the phenotypes would show the effect of the gene disruption. The temperature and medium phenotypes of wild type HB8 and mutant ΔTTHA 1897 was investigated to study the effect of the mutation in bacteria growth, and found to differ between the two strains in my thesis work.

2.2 Materials and Methods Bacto yeast extract and bacto tryptone were used as received from BD Bioscience (Sparks, MD). Ammonium chloride, Tri-Reagent, chloroform, phenol, 2-propanol, absolute ethanol and sodium chloride were purchased from Sigma-Aldrich (St Louis, MO). Ammonium acetate, potassium chloride and magnesium chloride were purchased from Fisher Scientific (Fairlawn, NJ). HPLC-grade methanol, water and acetonitrile were obtained from Honeywell Burdick & Jackson, Inc. (Muskegon, MI). Molecular biology grade Tris-hydrochloride was purchased from Promega (Madison, WI). Sodium citrate was purchased from Mallinckrodt Baker, Inc. (Paris, KY). RNase T1 was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Bacterial alkaline phosphatase was obtained from Worthington Biochemical Corporation (Lakewood, NJ). Syber gold was purchased from Invitrogen (Eugene, OR). NucleoBond RNA/DNA anion exchange columns were purchased from Macherey Nagel (Bethlehem, PA). Nanopure water (18 MOhms) from a Barnstead (Dubuque, IA) nanopure system was autoclaved before use

23 in sample preparation. An Implen Nano-Photometer Pearl was purchased from Implen GmbH (Munich, Germany).

2.2.1 T. thermophilus HB8 Wild Type Thermus thermophilus is an extreme thermophile that grows at high temperature (50~85°C). T. thermophilus was originally isolated from a thermal vent environment in Japan by Tairo Oshima and Kazutomo Imahori(43). T. thermophilus has several strains; the wild type strains used in this research are HB8 from Japan. To culture T. thermophilus a modified 162 medium was used. Cells were cultured in a 72°C incubator for about 1-2 days to reach saturation, the growth chart is shown in a later paragraph.

2.2.2 The Construction of ΔTTHA1897 Disruption Mutant The mutant ΔTTHA1897 was created by Dr. Limbach. It is a putative GidA null mutant. ΔTTHA1897 was constructed using pUC18 as the cloning vector. The htk gene cassette, optimized for disruption of ΔTTHA1897, was cloned into the PstI and HindIII sites of pUC18 to create plasmid pPL4K3 (Figure 2.1) with E. coli XL1 Blue as the host for initial plasmid construction. Wild-type HB8 was transformed with pPL4K3. Plasmid selection was done using ampicillin or kanamycin. Clones were selected using kanamycin. Integration of the htk gene cassette into the genome of HB8 was confirmed by PCR, restriction mapping and DNA sequencing. Escherichia coli XL1 Blue was cultured at 37 °C to mid-log phase. Thermus thermophilus HB8 and the ΔTTHA1897 disruption strains were cultured at 72 °C for 1-2 days to stationary phase for tRNA purification.

Figure 2.1 Plasmid map for pPL4K3

2.2.3 Total tRNA Isolation Tri-reagent RNA isolation protocol was used for RNA extraction. After cells were harvested, they were treated with Tri-reagent, chloroform and phenol consecutively. Tri- Reagent combines phenol and guanidine thiocyanate dissolved in a mono-phase solution to facilitate the immediate and effective inhibition of RNase activity. Cells were lysed in Tri-reagent and the lysate was separated into aqueous and organic phases by the addition of chloroform and centrifugation. RNA precipitation was facilitated by the addition of isopropanol and centrifugation.

A nucleobond AXG column was utilized for isolation of intact tRNA from RNA. Nucleobond AX is a silica-based anion exchange resin. The nucleobond resin could bind to the negatively charged phosphate backbone of RNA at pH 6.3. By controlling the concentration of salt in the buffer, tRNA could efficiently be eluted and be collected.

A 5% Polyacrylamide Gel was utilized to visualize total RNA and transfer RNA. Samples were stained by Syber Gold. The tRNA isolation quality could be examined based on the data collected from above experiments.

The concentration of total tRNA isolated was determined by measuring UV absorbance at 260 nm on an Implen NanoPhotometer Pearl. RNA purity was determined from the A260/A280 ratio. A ratio between 1.8 and 2.1 indicates RNA is not contaminated by protein.

2.2.4 Growth Conditions Several growth curves were measured for the HB8 wt and ΔTTHA1897 strains. Temperature studies were conducted at 60°C, 65°C, 70°C, 75°C, 80°C, and 82°C. A -1 -2 -3 -4 -5 culture of each strain was serially diluted to OD 600 of 10 , 10 , 10 , 10 , 10 ; then spotted onto plates containing TEM rich medium and incubated (shown in Appendix I). The medium studies were done in both TEM rich and minimal media at 72°C. Strains were cultured in 20 mL TEM at 72°C as a starter culture. After adjusting the OD600 of both starters to 0.05 with TEM, 10 mL of starters was added to start 1 L cultures in TEM and minimal Medium at 72°C.

2.3 Results and Discussion

2.3.1 Total tRNA Isolation tRNA from both HB8 wt and ΔTTHA1897 strains was isolated as described Figure 2.2 is a typical polyacrylamide gel image of total tRNA. The left lane is the total RNA from the cell, and the right lane is the tRNA purified using the NucleoBond column.

Figure 2.2. Total RNA (left) and tRNA (right) from T. thermophilus HB8

tRNA from both wild type and ΔTTHA1897 strains were isolated. Because of the similar number of nucleotides in 5S rRNA and tRNA, the isolation of tRNA from total RNA with the anion exchange NucleoBond column has a small amount of 5S RNA contamination in the tRNA fraction. However, the identification of post-transcriptionally modified nucleosides should not be affected, as 5S rRNA in T. thermophilus does not contain any modifications(44).

2.3.2 Phenotypes

2.3.2.1 Temperature-Sensitive Phenotypes The mutant grew slightly slower than the wild type, especially at low (60°C) and high (82°C) temperatures. As shown in Figure 2.3, the mutation clearly has an effect on the growth of the cells.

Temp(°C) OD -1 -2 -3 -4 -5 Temp(°C) OD -1 -2 -3 -4 -5 600 10 10 10 10 10 600 10 10 10 10 10 60 Wt 75 Wt D D

65 Wt 80 Wt D D

70 Wt 82 Wt D D

Figure 2.3 Temperature sensitive phenotypes study

2.3.2.2 Medium-Sensitive Phenotypes The growth curve of HB8 wt and ΔTTHA1897 in Rich TEM medium was conducted at 72°C as shown in Figure 2.4.

3.5

OD 600nm 2.5

2 HB8 wt

D1897 1.5

0.5

0 0 5 10 15 20 Time/hour

Figure 2.4. Growth curve of HB8 and ΔTTHA1897 in TEM rich media

Based on the growth curve in the TEM rich medium, the growth rate of ΔTTHA1897 and the wild type are similar, has no significant difference.

With the minimal medium, only the wild type was able to grow. No sign of growth was observed for the mutant. After incubation for about a week, culture in minimal medium for the wild type got cloudy, while the culture in the minimal medium for ΔTTHA1897 was still clear as it was made. Upon saturation, the OD600 reading of HB8 wild type showed a value of 0.25.

2.3.3 Discussion The differences in growth of the two strains under low and high temperature conditions indicate the TTHA1897 gene in T. thermophilus HB8 could affect its growth. As shown in Figure 2.3, at every temperature tested, the mutant ΔTTHA1897 shows slower growth

29 than the wild type control. At extreme temperatures, 60°C, 80°C, and 82°C, the mutant ΔTTHA1897 was under stress. With the gene disruption, ΔTTHA1897 was not able to replicate as the wild type with lower start density. As a result, the gene disruption of bacteria T. thermophilus HB8 leads to growth delay under high and low thermal stress.

More than that, the survival of the bacteria in minimal media is clearly affected by the TTHA1897 gene as well. Only wild type was able to grow in minimal media; the mutant ΔTTHA1897 could not grow. These results demonstrate the gene disruption has an impact on the bacteria T. thermophilus HB8’s growth and survival under media stress.

To sum up, in T. thermophilus HB8 the putative gidA gene TTHA1897 affected the growth and temperature sensitivity of the bacteria in the positive way, and disruption of this gene negatively effect the growth under thermal and nutrient stress.

2.4 Conclusions Mutant ΔTTHA1897 was created by disruption of the gene TTHA1897 with a kanamycin resistant gene. The growth phenotype study shows that the growth rate of the wild type HB8 is not significantly different than the mutant ΔTTHA1897 in rich medium. The temperature studies demonstrated that the mutant strain was not able to survive as the spotting assays show at high temperatures of 80 °C and 82 °C or low temperature of 60 °C. The medium study also revealed that the mutant could not survive in minimal medium. The phenotype of the two strains at different temperatures and in minimal media demonstrated that the gene TTHA1897 serves a function in the growth of Thermus Thermophilus. In the next chapter, the influence of the mutation on tRNA modifications will be analyzed.

Chapter 3 Identification of tRNA Modifications in T. thermophilus and Discovery of the Fragmentation Pattern of xm5s2U Nucleosides Family

3.1 Introduction Because tRNA plays an important role in gene expression, there is a great interest in studying these modifications. LC-MS is the most powerful tool for identifying tRNA modifications, especially at the nucleoside level. After LC separation, nucleosides can be detected by UV and mass spectrometry. In this chapter, the modified nucleosides from total tRNA of Thermus Thermophilus wild type HB8 and ΔTTHA 1897 strains were identified.

3.2 Materials and Methods

3.2.1 Enzymatic Digestion Nuclease P1 (Sigma), venom phosphodiesterase (Worthington) and antarctic phosphatase (New England Biolabs) were used to digest intact tRNA to nucleosides. After heat denaturation at 100 °C, Nuclease P1 (Nuclease 5'-Nucleotidehydrolase, 3'- Phosphohydrolase) was added to hydrolyze both 3' and 5'-phosphodiester bonds in RNA without base specificity to mono- and oligonucleotides terminated by a 3'-phosphate. Venom phosphodiesterase, which successively hydrolyzes 3'-hydroxy-terminated oligonucleotides to 5'-mononucleotides, was then added. Antarctic phosphatase was then added to catalyze the removal of 5´ phosphate groups from mononucleotides to generate nucleosides(45). Figure 3.1 is an illustration of the process of digestion of RNA to nucleosides.

Figure 3.1. Scheme of the process of digestion of RNA to nucleosides

3.2.2 HPLC-UV-LC/MS Analysis of Nucleosides HPLC was performed using a Hitachi D-7000 HPLC system with a Phenomenex Synergi 4 µ hydro-RP 80A column (250×2.0mm, 4 µ) reversed-phase column. A diode array detector (Hitachi L-7455) was utilized for UV absorbance detection. The UV spectrometer was set at 260nm. The mobile phase consisted of 5 mM ammonium acetate (Sigma) buffer, adjusted to pH 5.3 by acetic acid (Fluka) (A) and 40% acetonitrile (Honeywell) (B). The gradient profile is listed in Table 3.1.

Table 3.1. Gradient profile for HPLC of nucleosides

Interval Time (min) Composition (%B)

Start End Start End

0.0 5.8 1 1

5.8 9.2 1 2

9.2 10.9 2 3

10.9 12.7 3 5

12.7 32.0 5 25

32.0 38.0 25 50

38.0 43.5 50 75

43.5 45.0 75 75

45.0 50.0 75 99

50.0 55.0 99 99

55.0 60.0 99 1

60.0 65.0 1 1

For direct LC-MS analysis of nucleosides, the HPLC described above was connected in- line with a UV flow cell and a Thermo LTQ-XL ion trap mass spectrometer by splitting the flow from the HPLC column. Nucleosides were detected in positive polarity using an electrospray ionization source. Typically, 60 µg of total tRNA nucleoside digest, prepared in 20 µL of 5 mM ammonium acetate buffer, was injected per analysis.

3.2.3 Mass Condition Mass spectra were recorded in positive polarity. The LTQ XL mass spectrometry conditions are listed in Table 3.2 below. Table3.2 LTQ XL mass spectrometry conditions capillary temperature 275 °C spray voltage 4.0 kV sheath gas 45 arb auxiliary gas 20 arb

sweep gas 5 arb

3.3 Results and Discussion

3.3.1 HPLC-UV-LCMS Analysis of Nucleosides The tRNA digests from both Thermus thermophilus strains were analyzed by UV-LC-MS. All the nucleosides were sequentially eluted off the C18 column and detected by UV and mass spectrometry.

The identification of nucleosides based upon the data compiled by McCloskey (46). The retention time of nucleosides varied slightly from run to run because of slight differences in the LC-MS/MS running conditions such as temperature, while the elution order of the nucleosides remained the same. Figures 3.2 and 3.3 are the UV chromatograms of nucleosides in total tRNA of T. thermophilus HB8 and ΔTTHA 1897, respectively.

6 i A

2 k C Figure 3.2 UV Chromatogram of nucleosides in T. thermophilus HB8 wild type at 260 nm.

Table 3.3 Identified Nucleosides from total tRNA of T. thermophilus HB8 analyzed by LC- UV-MS

+ + + + Peak# Symbol tr(min) MH BH2 Peak# Symbol tr(min) MH BH2 1 D 4.8 247 NA 15 s2U/s4U 26.8 261 129 2 Ψ 5.2 245 NA 16 Gm 28.9 298 152

3 C 7.0 244 112 17 m1G/m2G 29.2 298 166

4 mcm5U 8.0 316 184 18 m1G/m2G 30.0 298 166

5 U 10.6 245 113 19 A 31.4 268 136

6 m1A 11.4 282 150 20 m5s2U 31.8 275 143

7 mnm5s2U 13.7 304 172 21 t6A 34.2 413 281

8 m7G 19.8 298 166 22 m2A/m6A 38.5 282 150

9 Cm 19.9 258 112 23 ms2t6A 40.8 459 327

10 I 23.1 269 137 24 io6A 43.9 352 218

5 6 11 m U 23.2 259 127 25 m 2A 44.0 296 164 12 G 24.4 284 152 26 ms2io6A 49.2 398 266

13 Um 26.3 259 113 27 i6A 50.0 336 204

14 k2C 26.8 372 240 28 ms2i6A 58.3 382 250

As seen in Table 3.3, a total of 28 nucleosides were identified through LC-UV-MS, including mnm5s2U.

Figure 3.3. UV Chromatogram of nucleosides in total tRNA of T. thermophilus ΔTTHA1897 at 260 nm

Table 3.4 Identified Nucleosides from total tRNA of ΔTTHA1897

+ + + + Peak# Symbol tr(min) MH BH2 Peak# Symbol tr(min) MH BH2 1 D 5.3 247 115 14 Gm 30 298 152

2 Ψ 5.9 245 15 m1G/m2G 30.1 298 166

3 C 8.2 244 112 16 m1G/m2G 31 298 166

4 mcm5U 9.4 316 184 17 A 32.4 268 136

5 U 12.6 245 113 18 m5s2U 32.8 275 143

6 m1A 13.9 282 150 19 t6A 35 413 281

7 Cm 22.3 258 112 20 m2A/m6A 39.6 282 150

8 m7G 22.2 298 166 21 ms2t6A 41.3 459 327

5 3 6 9 m U/m U 24.5 259 127 22 m 2A 44.5 296 164 10 G 25.7 284 152 23 io6A 44.4 352 220

11 Um 27.4 259 113 24 ms2io6A 49.7 398 266 12 k2C 27.6 372 240 25 i6A 50.6 336 204

13 s2U/s4U 28 261 129 26 ms2i6A 59 382 250

As seen in Table 3.4, 26 nucleosides have been identified in the T. thermophilus total tRNA of ΔTTHA1897 mutant strain, none of the xm5s2U nucleosides were identified.

3.3.2. Discussion In T. Thermophilus HB8, mnm5s2U and Inosine were identified. ΔTTHA1897 has a disrupted gene TTHA1897, which is a putative GidA gene found in E. coli to be essential for the biosynthesis of xm5s2U suggesting the removal of xm5s2U in ΔTTHA 1897 mutant was possible. Based on the results of the HPLC-UV-MS, none of the xm5s2U nucleosides were identified in the ΔTTHA1897 mutant nor was Inosine. Nevertheless, it does not rule out the possibility of instrument sensitivity that caused xm5s2U to be undetected. A more sensitive method may be need to be developed and applied.

3.4 Conclusions A total of 28 nucleosides in HB8 wild type tRNA were identified, including mnm5s2U; 26 nucleosides in ΔTTHA1897 were identified. Under the same conditions, mnm5s2U was not identified in ΔTTHA1897. Due to the low abundance of xm5s2U in the tRNA, the identification by the traditional LC-MS method was challenging.

Chapter 4. The Fragmentation Pattern of xm5s2U Nucleosides

4.1 Introduction The modified nucleosides in the xm5s2U family are relatively low in abundance when analyzed by LC-MS, causing great difficulties in xm5s2U identification. In this chapter, the fragmentation pathway of the xm5s2U family of nucleosides was determined, which contributed in the identification process.

4.2 Materials and Methods

4.2.1 Materials The chemicals used are the same as stated in Chapters 2 and 3.

4.2.2 HPLC-UV-LCMS Analysis of xm5s2U Standards The instrumental set up for the HPLC system and mass spectrometer is the same as stated in Chapter 3. The gradient profile and column are unchanged but the study subject chosen was a mixture of cmnm5s2U, mnm5s2U and nm5s2U nucleosides.

4.2.3 Direct Infusion Electrospray Mass Spectrometry The nucleosides standards cmnm5s2U, mnm5s2U and nm5s2U were generous gifts from Professor Jim McCloskey and Pam Crain, University of Utah. Standards were directly infused into the Thermo LTQ-FTTM with ESI source running at positive polarity. Samples were diluted in 5 mM ammonium acetate, pH 5.3. The flow rate of the syringe pump was set as 3 uL/min.

Table 4.1 LTQ-FT Mass spectrometry conditions

capillary temperature 275 oC capillary voltage 30 V sheath gas flow rate 14 aux gas flow 0 sweep gas flow 0 source voltage 5.0 kV tube lens voltage 69.77 V

The mass range was m/z 70 to 550. For tandem mass spectrometry, collision-induced dissociation (CID) was used in data-dependent mode. The data-dependent scan triggered fragmentation on the most abundant precursor ion. MS3 experiments were conducted on each molecule.

4.3 Results and Discussion

4.3.1 LC-MS Analysis of xm5s2U Standards Since cmnm5s2U and nm5s2U have yet to be detected in T. Thermophilus, the three standards were analyzed at the same LC-MS settings with a Phenomenex column. Figure 4.1 is the UV chromatogram of the standards. Table 4.2 is the amount of sample loaded and respective retention time of each nucleoside. Through MS/MS experiments, the fragmentation pattern was revealed, which trigged the direct infusion study on the FT-ICR mass spectrometer.

5 2 nm s U

5 2 mnm s U

5 2 cmnm s U

Figure 4.1 UV Chromatogram of xm5s2U standard at 260nm

Table 4.2 xm5s2U injection amount and elution time

+ + Standards Injected amount tr(min) MH BH2

nm5s2U 2 ug 10.3 290 158 mnm5s2U 2 ug 14.2 304 172 cmnm5s2U 0.6 ug 16.8 348 216

4.3.2 Direct Infusion of xm5s2U Standard on FT-ICRMS Standards of cmnm5s2U, mnm5s2U and nm5s2U were analyzed by FT-ICR MS and MS/MS, which yielded the accurate mass and elemental composition of the precursor and product ions. MS3 experiments were conducted on each nucleoside as well to assign the origin of each fragment ion.

4.3.2.1 Fragmentation of mnm5s2U

5 2 Figure 4.2 chemical structure of mnm s U (C11H17N3O5S)

The CID spectrum of mnm5s2U is shown in Figure 4.3. From MS/MS and MS3 experiments, the protonated mnm5s2U (m/z 304) generated the following fragment ions: m/z 273, 255, 237, 141, and 172 (the base ion). The m/z 273 ion generated m/z 255, and 141; the m/z 255 ion generated m/z 237 and 141. Accurate mass and corresponding elemental formula of these ions are shown in Table 4.3. These data are consistent with mnm5s2U fragmenting as illustrated in Figure 4.4.

mnm5s2U_052212_120521153620 #125-155 RT: 3.68-4.59 AV: 31 NL: 1.46E6 T: FTMS + p ESI Full ms2 [email protected] [80.00-550.00] 172.05381 100 95 90 255.04327 85 273.05383 80 75 70 65 60 55 50 45 40 RelativeAbundance 35 30 237.03274 25 20 225.03274 15 10 141.01164 161.55300 5 147.16459 181.74772 199.27150 220.74738 248.43017 268.07492 286.08549 0 140 160 180 200 220 240 260 280 m/z

Figure 4.3. The CID spectra of the precursor ion C11H18N3O5S (304)

Table 4.3. The accurate mass and the elemental formula of fragment ions from mnm5s2U

Calculated exact Mass Measured Mass Formula Delta (millimass unit)

304.09672 304.09604 C11H18N3O5S -0.128

273.05452 273.05383 C10H13O5N2S -0.139

255.04396 255.04327 C10H11O4N2S -0.134

237.03339 237.03274 C10H9O3N2S -0.099

141.01226 141.01164 C5H5ON2S -0.070

172.05446 172.05381 C6H10ON3S -0.099

Figure 4.4 The proposed fragmentation pathway of mnm5s2U

+ As shown in Figure 4.4, the positively charged molecular ion C11H18N3O5S m/z 304 lost + the neutral sugar ring m/z 132 and formed a positively charged base ion C6H10ON3S m/z

172. The molecular ion easily loses the neutral NH2CH3 (m/z 37) to form a relatively + stable ion C10H13O5N2S with m/z 273. The 273 ion could lose the sugar ring of m/z 132 forming a positively charged base ion m/z 141, and could lose a water of m/z 18 giving + + ion C10H11O4N2S m/z 255. The positively charged m/z 255 ion C10H11O4N2S lost the + + sugar ring to generate m/z 141 ion C5H5ON2S , and lost a water to produce C10H9O3N2S of m/z 237. In sum, there were three types of fragmentation throughout, one was the loss of sugar fragment as occurred in the transition of 304->172, 273->141, and 255-

>141; the other type was the loss of NH2CH3 in the transition of 304->273; the last type was the loss of water in the transition of 273->255, and 255->237.

4.3.3.2 Fragmentation of cmnm5s2U

5 2 Figure 4.5 Chemical structure of cmnm s U(C12H17N3O7S)

The CID spectrum of cmnm5s2U is shown in Figure 4.6. From MS/MS and MS3 experiments, cmnm5s2U (m/z 348) generated the following fragment ions: m/z 273, 255, 237, 141, and 216 (the base ion). The m/z 273 ion produced m/z 255, and 141. The m/z 255 ion formed m/z 237 and 141. Accurate mass measurement and corresponding elemental formula of these ions are shown in Table 4.4.

cmnm5s2U_2_052212_120521153620 #156 RT: 4.75 AV: 1 NL: 8.31E5 T: FTMS + p ESI Full ms2 [email protected] [95.00-550.00] 273.05386 100 95 90 85 255.04329 80 75 70 65 248.54713 60 55 50 45 40 RelativeAbundance 35 30 237.03278 25 216.04367 20 225.03278 15 127.52672 10 141.01164 5 153.01164 181.75404 208.08151 289.05322 312.06482 0 140 160 180 200 220 240 260 280 300 m/z

Figure 4.6 The CID spectra of the precursor ion C12H18O7N3S (348) Table 4.4 The mass list of ions from cmnm5s2U

Calculated exact Mass Measured Mass Formula Delta (millimass unit)

348.08655 348.08588 C12H18O7N3S -0.117

273.05452 273.05386 C10H13O5N2S -0.109

255.04396 255.04329 C10H11O4N2S -0.114

237.03339 237.03278 C10H9O3N2S -0.059

141.01226 141.01164 C5H5ON2S -0.070

216.04429 216.04367 C7H10O3N3S -0.068

Based on this data, the following fragmentation scheme of cmnm5s2U is proposed in Figure 4.7:

Figure 4.7 Proposed fragmentation pathway of cmnm5s2U

The fragmentation of cmnm5s2U and mnm5s2U has the same types of loss：the loss of sugar as occurred in the transition of 348->216, 273->141, and 255->141; the other was the loss of NH2CH2COOH (m/z 132) to form the relatively stable m/z 273 ion in the transition of 348->273; the last type was the loss of water in the transition of 273->255, and 255->237.

4.3.3.3 Fragmentation of nm5S2U

Figure 4.8 Chemical structure of nm5s2U

The CID spectrum of nm5s2U is shown in Figure 4.9. With MS/MS and MS3 experiments, the positively charged molecular ion of nm5s2U (m/z 290) generated fragment ions: m/z of 273, 255, 237, 141 and 216 (the base ion); the m/z 273 ion produced m/z 255 and 141 ions; the m/z 255 ion gave 237 and 141 ions. By using the FT-ICR mass spectrometer, the accurate mass and the corresponding formula of the ions were obtained as shown in Table 4.5.

Table 4.5 The mass list of ions from nm5s2U

Calculated exact Mass Measured Mass Formula Delta (millimass unit)

290.08107 290.08041 C10H16O5N3S -0.108

273.05452 273.05387 C10H13O5N2S -0.099

255.04396 255.04330 C10H11O4N2S -0.104

237.03339 237.03279 C10H9O3N2S -0.049

141.01226 141.01165 C5H5ON2S -0.060

158.03881 158.03821 C5H8ON3S -0.049

nm5s2U_052212_120521153620 #123-220 RT: 3.60-6.57 AV: 98 NL: 2.54E6 T: FTMS + p ESI Full ms2 [email protected] [75.00-550.00] 273.05387 100 95 90 85 80 75 70 65 60 255.04330 55 50 45 40 RelativeAbundance 35 30 25 20 15 237.03279 10 225.03279 5 136.52736 280.11193 158.03821 181.75121 194.99739 220.74577 247.09503 266.67666 291.08386 0 140 160 180 200 220 240 260 280 m/z

Figure 4.9 The CID spectra of the precursor ion C10H16O5N3S (290)

Based on data collected shown in above discussion, the fragmentation of nm5s2U is proposed in Figure 4.10:

Figure 4.10. Proposed fragmentation pathway of nm5s2U.

The fragmentation of nm5s2U and mnm5s2U has the same types of loss：the loss of sugar in the transition of 290->1158, 273->141, and 255->141; the other type was the loss of functional group NH3 (m/z 17) to form the relatively stable 273 ion in the transition of 290->273; the last type was the loss of water molecule in the transition of 273->255, 255->237.

4.3.3.4 The Proposed Fragmentation Pathway of xm5s2U As seen in previous discussion, the xm5s2U family of nucleosides gave the same fragmentation pathway; mass differences occurred due to the loss of different functional groups at the 5 position of the purine ring. We have used mnm5s2U as an example to illustrate the fragmentation of these types of molecules. The proposed fragmentation pathway of xm5s2U is shown in Figure 4.11:

5 2 xm s U ——>C10H13O5N2S ——> C10H11O4N2S——>C10H9O3N2S (m/z 273) (m/z 255) (m/z 237)

Protonated base ion C5H5ON2S (m/z 141)

Figure 4.11 Schemed illustration of xm5s2U fragmentation pathway.

4.4 Conclusions Compared with other nucleosides in which a loss of sugar ring is the typical result under CID, The xm5s2U family of nucleosides has a unique fragmentation pattern under collision induced dissociation. By taking advantage of FT-ICR MS, the accurate mass of all fragments was measured revealing the molecular formula. The fragmentation pattern of xm5s2U is proposed in Figure 4.11. The xm5s2U family of nucleosides tends to lose sugar, the functional group on the 5 position of the pyrimidine ring, and water molecules. As the unique fragmentation pattern is revealed, methods could be developed for better detection of xm5s2U nucleosides. The next chapter is about the application of these fragmentation patterns and the development of mass spectrometry-based methods in the detection of xm5s2U nucleosides.

Chapter 5 SRM method for the study of xm5s2U family nucleosides

5.1 Introduction Due to the low abundance nature of xm5s2U nucleosides in Thermus thermophilus, a better detection MS method is needed. As the unique fragmentation pattern has been revealed in Chapter 4, the Selected Reaction Monitoring (SRM) method was used aiming for better detection of xm5s2U nucleosides.

5.2 HPLC-UV-MS Instrumental Conditions The HPLC-UV-MS system was set up the same way as previous mentioned in Chapter 3. The only difference here is the HPLC column is a Supeleco LC-18-S column (250mm Х 2.0mm, 4µ). HPLC was running the same gradient coupled with UV detector. Mass spectrometer has an ESI source, after LC separation, analytes were analyzed in the mass spectrometer in positive mode. The mass spectrometer conditions are listed in Table 5.1. The sample used was E. coli tRNA (Sigma) digested as stated in Chapter 3.

Table 5.1 LTQ-FT Mass spectrometry conditions

capillary temperature 275 C capillary voltage 13 V sheath gas flow rate 25 aux gas flow 14 sweep gas flow 10 source voltage 4.0 kV tube lens voltage 70 V

The mass range was m/z 100-600, sample used was 60 μg of E. coli total tRNA nucleoside digests. Activation type was Collision Induced Dissociation (CID), normalized collision energy was 35%, Q value was 0.25, scan time was 30 ms, isolation width was 2 m/z. The following reactions were monitored: nm5s2U 290-> 158, 255, 273; mnm5s2U

304-> 172, 255, 273; cmnm5s2U 348-> 216, 255, 273. Data dependent acquisition was enabled during the run.

5.3 Result and Discussion As standard E. coli tRNA nucleoside digests were used, the SRM method demonstrated its effectiveness in identifying xm5s2U. cmnm5s2U (MH+ 348) and mnm5s2U (MH+ 304) were detected around 15 and 20 minutes respectively, as shown in Figure 5.1. For comparison purpose, those ions and their base ions were not detected in MS scans.

Figure 5.1 the ion chromatogram of cmnm5s2U and mnm5s2U of SRM scans

Figure 5.2 the CID mass spectrum of the monitored reaction of cmnm5s2U

Figure 5.3 the CID mass spectrum of the monitored reaction of mnm5s2U.

As shown in Figures 5.1, 5.2, 5.3, with the SRM method, the mass spectrometer was able to detect cmnm5s2U at retention time between 15-17 minutes, and mnm5s2U at around 20-22 minutes. The mass spectrum of cmn5s2U and mnm5s2U demonstrated the unique fragmentation pattern of both, with their own base ion and unique fragments of 237, 255 and 273 ions detected. The transition to 141 fragment ion was not listed for detection in this SRM method. Comparing with the experiments with only data dependant mode, SRM method apparently improved the detection of cmn5s2U.

5.4 Conclusion An effective SRM method for detection of xm5s2U nucleosides was developed and demonstrated by the detection of cmnm5s2U and mnm5s2U in E. coli tRNA digest. In the future, this SRM method could be used for the detection of xm5s2U in Thermus Thermophilus HB8 ΔTTHA1897 strain for verifying the existence of xm5s2U.

Chapter 6 Neutral loss on Triple quadrupole mass spectrometer

6.1 Introduction As the fragmentation pattern is revealed, we would want to apply it to set up a neutral loss method on a triple quadrupole mass spectrometer for the detection of the xm5s2U family of nucleosides. The original thought is that the best instrument to do true neutral loss experiment would be triple quadrupole mass spectrometer. The mass spectrometer was purchased from Waters in 1990s, which has some sensitivity issue based on the usage experience when conducting the following experiments described in this chapter.

6.2 Experimental

6.2.1 Materials and Instrument Conditions ESI/MS analyses were performed on Triple Quadrupole mass spectrometer Micromass Quattro LC from waters equipped with an ESI source and controlled by MassLynx Software. E. coli total tRNA nucleosides digest (Sigma) was separated by the same HPLC system as described in Chapter 5. The HPLC column used was a Supeleco LC-18-S column (250mm Х 2.0mm, 4µ). The mass spectrometer conditions are listed in Table 6.1. The sample injection amount was increased to 160 ug of tRNA digests to increase the possibility of detecting xm5s2U.

Table 6.1 Triple Quadrupole mass spectrometer conditions

Source temperature 100 °C Capillary voltage 2.8 kV Sample cone voltage 18 kV Q1 Low mass resolution 1 18 eV high mass resolution 1 15 eV Ion Energy 1 0.8 eV q Entrance 42 eV collision energy 15 eV for experiment 1 40 eV for experiment 2 exit 90 eV Q2 Low mass resolution 2 16 eV high mass resolution 2 17 eV Ion Energy 2 2.5 eV Multiplier voltage 685 V

6.2.2 MRM and Neutral loss Scan Set Up Ions of interest were selected in the first quadrupole and accelerated into a collision cell containing argon to induce CID. Product ions were then analyzed in the final quadrupole. Based on the fragmentation pattern of xm5s2U discovered in Chapter 4, the transitions and neutral loss were designed. Table 6.2 is the proposed fragmentation pattern of xm5s2U displayed in m/z manner. The corresponding chemical formula is documented in Chapter 4.

Table 6.2 is the fragmentation pattern of xm5s2U displayed in m/z manner

5 2 cmnm s U 348 -NH2CH2COOH(75) 273 -H2O(18) 255 -H2O(18) 237

C12H18O7N3S C10H13O5N2S C10H11O4N2S C10H9O3N2S

216 141

C7H10O3N3S C5H5ON2S 5 2 mnm s U 304 -NH2CH3(31) 273 -H2O (18) 255 -H2O(18) 237

C11H18N3O5S C10H13O5N2S C10H11O4N2S C10H9O3N2S

172 141

C6H10ON3S C5H5ON2S 5 2 nm s U 290 -NH3(17) 273 -H2O(18) 255 -H2O(18) 237

C10H16O5N3S C10H13O5N2S C10H11O4N2S C10H9O3N2S

158 141

C5H8ON3S C5H5ON2S

Experiment 1 has 7 scan events, including an MS scan for the mass range of 100 to 700 and 3 MRM scans for 3 nucleosides, monitoring the transitions: cmnm5s2U (348—>273 transition), mnm5s2U (304—>273 transition) and nm5s2U (290—>273 transition), plus 3 neutral loss scans of the three nucleosides, which are 75, 31 and 17, as listed in Table 6.3. Table 6.3 The scan events in experiment 1.

Experiment 1

MRM scans Neutral loss scans MS scan

348—>273 transition; 75 of 348; mass range: 100 - 700 304—>273 transition; 31 of 304; 290—>273 transition 17 of 290

Experiment 2 has 10 scan events. Including the 3 same MRM scans as experiment 1 monitoring the transitions: cmnm5s2U (348—>273 transition), mnm5s2U (304—>273 transition) and nm5s2U (290—>273 transition); three new MRM scans of cmnm5s2U (348->216 transition), mnm5s2U (304->172 transition), nm5s2U (290->158 transition) (loss of 132 for all), experiment 2 has the 3 neutral loss scans of the three nucleosides: 75, 31 and 17, and an extra neutral loss scan of 132, which is the base loss for all nucleosides. Experiment 2 did not have the MS scan as listed in Table 6.4.

Table 6.4 The scan events in experiment 2

Experiment 2

MRM scans Neutral loss scans MS scan

348—>273 transition; 132 for all nucleosides; NA 348->216 transition； 75 for 348; 304—>273 transition; 31 for 304; 304->172 transition； 17 for 290. 290—>273 transition； 290->158 transition.

6.3 Results and discussion

6.3.1 Experiment 1: MS Scan, MRM and Neutral Loss Scans The ability of this triple quadrupole mass spectrometer was unknown for MS scan, MRM scans and neutral loss scans. Unfortunately, the neutral loss method did not work even though the MRM method worked. In experiment 2, another neutral loss method was implemented to further test the correctness of the neutral loss settings.

6.3.1.1 MS Scan Since this triple quadrupole instrument was not previously used in our lab, the nucleosides of E. coli tRNA detected was listed and compared with LTQ result in Table 6.4 to demonstrate the sensitivity of the instrument. It is necessary to point out the difference in sample loading of 60 ug tRNA on the LTQ vs. 160 ug tRNA on the triple quadrupole.

Table 6.4 Modified nucleosides of E. coli total tRNA in experiment 1 number of nucleosides Nucleosides t (min) MH+ detected on LTQ r 1 D 4.44 247 2 ψ 3.92 245 3 C 6.12 244 4 cmo5U 4.03 319 5 acp3U 8.4 346 6 U 8.4-8.7 245 7 s2C 10.2 260 8 mnm5s2U 20.2 304 9 m7G 18.37 298 10 Cm 19.4 258 11 I 20.7 269 12 m5U 20.47 259 13 G 21.93 284 14 Um 23.82 259 15 s4U 22.56 261 16 m4Cm/m5Cm NA 272 17 k2C NA 372 18 Gm 24.13 298 19 m2G 25.07 298 20 m1G 26.5 298 21 ac4C NA 286 22 A 28.42 268 23 t6A 30.41 413 24 m2A 35.34 282 25 m6A 35.86 282 26 m6t6A 34.8 427 27 ms2t6A NA 459 28 m6,2A 43.08 296 29 i6A 49.89 336 30 ms2i6A 58.5 382 31 Q 45.6 410 *NA: not detected on Triple Quadrupole mass spectrometer

As shown in Table 6.4, the triple quadrupole could only detect 27 nucleosides despite more than two times of sample loaded due to sensitivity issue for this particular mass spectrometer. In comparison, 31 nucleosides were successfully detected by an LTQ mass analyzer in previous experiments.

6.3.1.2 Detection of mnm5s2U with MRM Scan and Neutral Loss Scan In experiment 1, mnm5s2U (304) could be detected in the MRM scans, but not in neutral loss scans. Figure 6.1 is the extracted ion chromatograms of 304. Because the m/z of the sodium adduct of m1A (282) is 304 as well, it is necessary to lay out the chromatogram of 282 together with 304. As shown in Figure 6.2, the retention times of the two on the Supeleco column were far apart, suggesting that 304 peak was not related to the sodium adduct of 282. No meaningful information was obtained for cmnm5s2U and nm5s2U, as shown in Figure 6.3 and 6.4.

ECOLI 071113_ECOLI_TRNA_lihua 3: Neutral Loss 31ES+ 64.17 100 304 0.5000Da 26.47 50.76 90 12.33 32.02 38.83 39.77 46.99 52.33 58.41 2.81 4.90 8.88 14.01 17.88 25.74 64.79 55.68

% 21.86

0 10.00 20.00 30.00 40.00 50.00 60.00 70.00 071113_ECOLI_TRNA_lihua 5: MRM of 3 Channels ES+ 20.64 100 304 > 273 20.12 48.08 770 33.10 37.08 38.65 40.01 50.80 54.99 59.38 64.51 3.36 10.38 18.13 26.19 7.66 12.89 %

0 10.00 20.00 30.00 40.00 50.00 60.00 70.00 071113_ECOLI_TRNA_lihua 7: MRM of 1 Channel ES+ 20.54 100 304 > 273 60.1363.37 780 19.81 21.17 30.60 42.85 44.11 6.09 33.11 39.60 48.19 57.61 64.42 0.34 11.44

% 4.63

0 10.00 20.00 30.00 40.00 50.00 60.00 70.00 071113_ECOLI_TRNA_lihua 1: Scan ES+ 20.26 100 303.297_304.8 0.5000Da 20.47 1.51e3 5.81 19.94 % 6.64 22.77 66.91 69.79 26.33 41.09 61.93 5.07 11.88 16.28 32.40 37.64 47.59 56.70 66.07 50.94 69.88 0 Time 10.00 20.00 30.00 40.00 50.00 60.00 70.00

Figure 6.1 neutral loss of 31 scan, MRM of 304->273, and XIC of 304 (mnm5s2U) In experiment 1

ECOLI 071113_ECOLI_TRNA_lihua 1: Scan ES+ 35.34 100 281.5_282.8 0.5000Da 9.22e3 % 35.86

36.38

39.63 40.57 21.62 29.99 33.03 0 20.00 25.00 30.00 35.00 40.00 071113_ECOLI_TRNA_lihua 1: Scan ES+ 20.26 100 303.297_304.8 0.5000Da 1.51e3

20.47

20.78

21.41 % 19.94

22.77 41.09 37.64 23.71 32.40 37.01 16.28 40.57 18.27 25.39 26.33 27.69 34.71 42.66 28.8430.62 39.63

0 Time 20.00 25.00 30.00 35.00 40.00

Figure 6.2 XIC of 282 (m1A) and 304 (mnm5s2U) in Experiment 1

As we can see from Figure 6.1, the MRM scan showed better sensitivity in Experiment 1 for detection of mnm5s2U. Figure 6.2 shows that m1A (282) has a retention time of 35.3 minutes, whereas mnm5s2U (304) has a retention time of 20.3 minutes which means the peak of 304 is not the sodium adduct of 282. Therefore, it is safe to say that the MRM method on the triple quadrupole is successful for the detection of mnm5s2U.

ECOLI 071113_ECOLI_TRNA_lihua 6: MRM of 1 Channel ES+ 64.62 348 > 273 100 33.11 41.27 46.82 50.7055.30 57.61 60.54 23.47 37.50 542 18.03 19.81 29.02 8.81 13.21 0.65 6.09 8.29 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua 5: MRM of 3 Channels ES+ 37.19 63.57 348 > 273 100 45.4648.08 52.47 62.21 64.62 43.05 49.54 53.73 56.45 617 19.49 25.35 30.80 34.46 6.09 12.6814.04 23.36 27.66 4.41 7.45 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua 2: Neutral Loss 75ES+ 11.37 31.79 55.67 348 0.5000Da 100 38.91 40.69 59.54 24.36 52.42 61.32 81 3.10 46.45 49.28 4.99 13.68 17.45 20.69 58.49 8.23 35.25 45.30 26.2426.87 38.50 63.21 2.79 63.83 % 9.17

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua 1: Scan ES+ 4.13 100 348 0.5000Da 312 41.51 47.17 8.11 36.28 6.22 15.02 2.04 46.85 54.08 57.43 58.27 8.5313.76 21.09 52.93 65.74 % 18.58 67.34 30.20 32.51 44.86 55.96 62.04 9.89 38.37 69.40 27.80 63.71 0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00

Figure 6.3 MRM of 348->273, neutral loss of 75, and XIC of 348 (cmnm5s2U) in experiment 1.

ECOLI 071113_ECOLI_TRNA_lihua 8: MRM of 1 Channel ES+ 46.83 49.97 60.55 64.63 290 > 273 100 37.20 41.18 45.26 53.64 56.57 31.86 33.01 537 10.18 15.73 23.48 26.83 11.44 19.50 20.44 1.81 4.74 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua 5: MRM of 3 Channels ES+ 64.83 290 > 273 100 30.80 38.55 49.54 57.71 59.28 34.46 37.40 43.05 44.10 52.16 548 9.12 18.44 20.75 23.78 28.91 5.98 12.58 4.62 8.70 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua 4: Neutral Loss 17ES+ 30.78 100 290 0.5000Da 31.31 64.60 94 23.14 26.91 38.95 42.72 47.43 52.46 56.75 58.95 12.98 17.38 25.55 37.07 1.15 10.05 56.54 4.716.80 27.96 43.66 21.36 % 4.39

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua 1: Scan ES+ 8.42 100 290 0.5000Da 8.84 900 28.63 8.21 28.21 30.10 35.65 % 7.48 44.97 54.71 56.38 65.81 10.20 24.76 42.77 50.41 59.42 2.044.03 13.24 39.21 62.56 66.43 17.85 20.78 69.60 0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00

Figure 6.4 MRM of 290->273, neutral loss of 17, and XIC of 290 (nm5s2U) In experiment 1.

Combining the information drawn from Figure 6.3 and Figure 6.4, cmnm5s2U and nm5s2U were not detected in this experiment with any method.

6.3.2 Experiment 2: MRM and Neutral loss scans without MS scan In Experiment 2, the collision energy was increased to 40 eV and MS scan was not used to focus on MS/MS scans. This experiment was meant to test whether the neutral loss settings were set up correctly and sensitive enough for the detection of xm5s2U nucleosides. The TIC of neutral loss of 132 scan in experiment 2 is shown in Figure 6.5. Some high abundance nucleosides were detected in the chromatogram indicating that the method or the instrumental set up for this method was correct.

ECOLI 071113_ECOLI_TRNA_lihua02_no_ms_scan 8: Neutral Loss 132ES+ 20.81 100 TIC 6.23e4

5.50

28.21 % 7.43

18.58 23.85 29.94

58.43 3.98 34.19

0 Time 10.00 20.00 30.00 40.00 50.00 60.00 70.00

Figure 6.5 Total Ion Chromatogram of neutral loss of 132 in experiment 2

6.3.2.1 Neutral loss scans comparison for cmnm5s2U (348), mnm5s2U (304), nm5s2U (290) To compare the effectiveness of the neutral loss scans for each particular nucleoside, the neutral loss of cmnm5s2U (348), mnm5s2U (304), nm5s2U (290), 75, 31, 17 and 132 were listed in Figure 6.6. No xm5s2U nucleoside was detectable in any of those scans. The neutral loss scans were not sensitive enough for the detection of cmnm5s2U, mnm5s2U, or nm5s2U.

ECOLI 071113_ECOLI_TRNA_lihua02_no_ms_scan 8: Neutral Loss 132ES+ 5.60 40.58 42.31 290 0.5000Da 100 2.15 8.95 14.42 17.67 22.74 29.12 32.1732.88 54.17 55.89 62.99 4.49 10.47 25.07 51.43 58.73 46.16 64.51 77 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 8: Neutral Loss 132ES+ 17.67 304 0.5000Da 100 14.52 13.61 20.51 235 4.59 20.81 27.81 29.33 % 13.10 2.66 8.75 24.76 33.59 38.25 41.70 46.16 50.8251.53 56.30 59.24 63.29 64.21 0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 8: Neutral Loss 132ES+ 37.54 100 7.02 9.05 348 0.5000Da 17.67 32.88 36.42 45.65 50.01 60.15 63.19 102 1.85 6.21 10.77 18.38 23.24 27.60 39.57 48.08 53.97 57.72 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 3: Neutral Loss 17ES+ 49.66 100 290 0.5000Da 24.92 46.42 137 1.70 6.77 12.35 14.07 19.95 51.69 57.77 62.03 63.85 6.37 7.79 22.69 30.09 36.58 43.58 % 29.08 31.00 42.77 0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 2: Neutral Loss 31ES+ 14.76 100 16.79 304 0.5000Da 21.15 91 12.33 26.42 32.61 36.56 39.7144.98 57.65 62.11 1.89 4.42 9.49 30.07 34.23 47.11 49.95 53.60 59.88

% 64.75

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 1: Neutral Loss 75ES+ 64.02 348 0.5000Da 100 4.71 7.14 14.04 22.55 29.95 40.20 42.73 58.65 61.99 8.05 15.76 24.99 44.86 52.56 64.43 67 2.38 32.49 36.04 %

0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00

Figure 6.6 The neutral loss chromatogram of 75, 31, 17 and 132 for cmnm5s2U (348), mnm5s2U (304), and nm5s2U (290) accordingly in experiment 2

6.3.2.2 Neutral loss and MRM scans comparison for cmnm5s2U, mnm5s2U, nm5s2U To test the effectiveness of this experimental setting between MRM and neutral loss method, the neutral loss of unique mass (75, 31, 17 accordingly), neutral loss of 132, MRM of unique transition (348->273, 304->273, 290->273), and MRM of sugar loss chromatograms for cmnm5s2U, mnm5s2U, nm5s2U, were lined up in Figures 6.7, 6.8 and 6.9 respectively. MRM methods showed better sensitivity than neutral loss method for this instrument.

ECOLI 071113_ECOLI_TRNA_lihua02_no_ms_scan 6: MRM of 1 Channel ES+ 18.66 100 16.83 19.27 304 > 172 14.50 2.34e3 13.39 19.98 12.58 % 10.04 26.26 5.28 21.90 30.42 33.06 44.72 50.09 53.13 62.97 2.23 7.91 24.64 27.38 37.42 40.46 42.99 51.51 57.39 64.39 0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 4: MRM of 3 Channels ES+ 14.90 17.53 42.78 58.80 304 > 273 100 3.75 13.58 23.52 26.05 29.40 48.76 60.22 19.46 27.47 31.43 37.51 38.62 41.56 52.2152.8257.08 62.86 573 3.24 7.80 10.54 4.86 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 8: Neutral Loss 132ES+ 17.67 100 304 0.5000Da 14.52 235 13.61 20.51 13.31

% 13.10 20.10 20.81 27.81 33.59 2.66 5.70 8.75 24.76 37.6438.25 41.70 46.16 50.82 51.53 4.59 29.33 56.30 59.24 63.29 64.21

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 2: Neutral Loss 31ES+ 14.76 100 16.79 304 0.5000Da 91 12.33 20.34 21.15 30.07 32.61 36.56 57.65 62.11 1.89 9.49 24.80 34.23 39.71 44.98 47.11 59.88 % 4.42 6.45 26.42 49.95 53.60 64.75

0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Figure 6.7 mnm5s2U neutral loss of 31, and neutral loss of 132, MRM of 304->273, and MRM of 304->172 chromatograms in Experiment 2

As shown in Figure 6.7, the MRM method focusing on the transition of sugar loss on mnm5s2U (304->172 transition) was detected, but transition 304->273 which was detected in experiment 1 was not detected here. In Figure 6.8 and Figure 6.9, none of the scans were able to detect cmn5s2U or nm5s2U.

ECOLI 071113_ECOLI_TRNA_lihua02_no_ms_scan 1: Neutral Loss 75ES+ 100 348 0.5000Da 4.71 7.14 29.95 40.20 67 14.04 42.73 44.86 52.56 58.65 61.18 61.99 6.64 8.05 15.76 24.3824.99 28.03 37.36 50.23 2.38 18.29 36.04 55.81 20.93 32.49 64.43 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 8: Neutral Loss 132ES+ 37.54 100 348 0.5000Da 7.02 102 33.89 36.42 6.21 9.05 17.67 50.01 1.85 9.35 27.60 45.6548.08 53.97 57.72 13.31 18.38 23.24 32.88 39.57 44.74 60.15 63.19 4.18 15.94 25.98 53.05 63.80 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 4: MRM of 3 Channels ES+ 2.12 3.64 51.70 59.92 63.26 348 > 273 100 7.90 13.68 16.22 20.68 23.31 24.94 38.93 43.49 47.24 53.22 55.96 6.69 14.90 30.31 33.35 40.35 566 1.01 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 5: MRM of 1 Channel ES+ 5.98 100 348 > 216 1.77e3

3.65 36.60

% 7.50 3.45 10.65 44.51 50.19 59.01 64.69 15.21 16.02 20.48 23.32 26.36 30.31 33.05 39.95 45.83 51.61 54.35 62.15 63.06

0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Figure 6.8 cmnm5s2U neutral loss of 75, and neutral loss of 132, MRM of 348-273, and MRM of 348-216 chromatograms in Experiment 2

ECOLI 071113_ECOLI_TRNA_lihua02_no_ms_scan 8: Neutral Loss 132ES+ 5.60 40.58 42.31 290 0.5000Da 100 2.15 17.67 29.12 51.43 54.17 55.89 8.95 14.42 32.17 32.88 36.73 42.81 77 10.47 22.74 25.07 46.16 58.73 63.80 4.49 11.38 1.04 20.81 48.08 64.51 % 5.91

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 3: Neutral Loss 17ES+ 49.66 100 290 0.5000Da 46.42 137 24.92 45.81 53.92 57.77 1.70 6.77 12.35 19.95 51.69 62.03 6.37 36.58 43.58 % 7.79 14.07 22.69 30.09 4.34 17.82 29.08 31.00 63.85 10.73 17.11 38.61 64.77

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 4: MRM of 3 Channels ES+ 34.26 100 290 > 273 34.57 52.82 55.86 752 45.92 54.44 60.93 4.96 6.38 6.79 13.38 16.12 20.58 24.43 25.24 31.73 40.45 47.24 64.78 3.54 9.12 39.03 %

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 071113_ECOLI_TRNA_lihua02_no_ms_scan 7: MRM of 1 Channel ES+ 48.58 57.50 59.02 290 > 158 100 20.29 25.96 28.60 30.12 53.24 8.93 18.77 37.32 40.46 44.42 50.50 63.07 587 2.44 3.56 7.92 14.71 17.65 34.48 %

0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Figure 6.9 nm5s2U neutral loss of 17, and neutral loss of 132, MRM of 290-273, and MRM of 290-158 chromatograms in Experiment 2

6.4 Conclusions Based on experiment 1 and 2, I propose that:

The collision energy of q2 was at 15 eV (as in experiment 1) which is better than 40 eV (as in experiment 2) for the unique reaction to generate 273 ions suggesting a reasonable explanation of the fact that MRM peak for transition 304->273 was not observed in experiment 2. Therefore, it might be helpful to use lower collision energy in further experiments.

Let the instrument do only 1 or 2 particular MRM/ neutral loss scan at each run might improve sensitivity, but at a high cost of sample consumption. The neutral loss scans were not as sensitive as MRM scan, but whether if it is related to the instrument itself or the settings is not clear yet.

Chapter 7 Conclusions and Future Work

7.1 Thesis Conclusions tRNA modifications are key factors of tRNA structure and function, especially in gene translation. The study of tRNA modifications has drawn a lot of attention in the past few decades. The science of tRNA modifications has made many achievements and allowed a better understanding. Still a lot of mysteries regarding tRNA modifications are unrevealed. The identification, function, structure and biosynthesis of tRNA modifications, the sequence of tRNAs, and the locations of the modification on tRNA are all interesting subjects waiting to be further explored.

In this work, Thermus thermophilus wild type and ΔTTHA 1897 strains were used to study the xm5s2U nucleosides, the existence in both wild type and ΔTTHA 1897, and the possible function of xm5s2U. In Chapter 2, tRNA was isolated from both strains, and digested for further HPLC-UV-MS study. Growth phenotype studies including growth rate and a temperature sensitivity study. Compared with wild type, the growth rate of

ΔTTHA1897 strain was smaller at the high end and low end of the temperature range, and a period of delay period was observed for the cell multiplication in minimal media.

The results demonstrated that ΔTTHA1897 was influenced by the mutation, which is the disruption of tRNA gene TTHA 1897 in Thermus thermophilus.

In Chapter 3, the tRNA nucleoside digests from both wild type and ΔTTHA 1897 were analyzed using HPLC-UV-MS. A total of 28 tRNA nucleosides were identified in the wild type including mnm5s2U and inosine. However a total of 26 tRNA nucleosides were identified in ΔTTHA1897, which was missing mnm5s2U and inosine.

In Chapter 4, taking advantage of FT-ICR MS, the accurate mass of all fragments was measured providing the molecular formula of each fragment. The fragmentation pattern of xm5s2U was proposed. Compared with other nucleosides in which loss of a sugar is the typical result under CID, the xm5s2U family of nucleosides tends to have a unique mass spectrum from loss of sugar, the functional group on the 5 position of the pyrimidine ring, and water. This unique fragmentation pattern could be used to develop more sensitive methods for better detection of xm5s2U nucleosides.

In Chapter 5, an effective SRM method was developed and tested using E. coli tRNA nucleosides and the results were briefly discussed. cmnm5s2U and mnm5s2U were both identified in E. coli tRNA nucleosides digest. In Chapter 6, a triple quadrupole mass spectrometer was used to examine a neutral loss method for the identification of xm5s2U nucleosides. After several trials, it was found that the neutral loss method did not have the same sensitivity as the MRM method on this instrument. Moreover, the collision energy is better at 15 eV than 40 eV to protect the unique fragmentation of xm5s2U family on this instrument, which might further contribute to the low detection abilities.

7.2 Future work As the discovery of the unique fragmentation pattern and the development of the SRM method for detection of xm5s2U, the first step would be to prepare ΔTTHA1897 tRNA, and then test the existence of xm5s2U with the SRM method. Quantification of xm5s2U

72 could be conducted after these steps. More experiments could be done on the triple quadrupole mass spectrometer with less scans per each run. If the sensitivity of the neutral loss runs improves, it could be beneficial to try the neutral loss method for the detection of xm5s2U using a triple quadrupole instrument.

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Appendix I Modified Minimal TEM for Thermus species (TMM)

Distilled water 860.0 ml

. Na3citrate 2H2O 154.0 mg

. CaSO4 2H2O 40.0 mg

. MgCl2 6H2O 200.0 mg

0.01 M Fe citrate 0.5 ml

Trace Element Solution 0.5 ml

Agar 28.0 g

Adjust pH to 7.2 with NaOH. Autoclave at 121oC for 15 minutes.

Autoclave the phosphate buffer, carbon source, and supplements separately and then add to the medium.

Phosphate Buffer 100 ml

20 % Sucrose 20 ml 0.4 % final

1M NH4Cl 10 ml 10 mM final

Vitamin solution A 10 ml

Other carbon sources (have only gotten sucrose to work thus far):

20 % Glucose 20 ml 0.4 % final

10 % glutamate 20 ml 0.2 % final

10 % Na acetate 20 ml 0.2 % final

For Thermus thermophilus HB8, also add lysine to 20 µg/ml.

10 mg/ml Lysine 2 ml

Vitamin Solution A (4 mg/l each, filter sterilize):

Paraminobenzoic acid

Nicotinic acid

Ca pantothenate

Thiamine

Pyridoxine

Biotin

Cyanocobalamine

Phosphate Buffer:

KH2PO4 5.44 g

Na2HPO4 17.0 g

Distilled water 1.0 L

Adjust pH to 7.2.

Trace Element Solution:

. Na3citrate 2H2O 19.7 g 1.97 g 9.85 g

. FeCl2 4H2O 1.0 g 100 mg 0.5 g

. MnCl2 4H2O 0.5 g 50 mg 0.25 g

. CoCl2 6H2O 0.3 g 30 mg 150 mg

. CuCl2 2H2O 50.0 mg 5 mg 25 mg

. Na2MoO4 2H2O 50.0 mg 5 mg 25 mg

H3BO3 20.0 mg 2 mg 10 mg

. NiCl2 6H2O 20.0 mg 2 mg 10 mg

Distilled water 1.0 L 100 ml 500 ml

Notes:

. 1. Na3citrate 2H2O replaces Nitrilotriacetic acid, which is a teratogen.

2. 0.01 M Fe citrate is made up as a 245 mg/100 ml solution in water. The

Fe citrate dissolves upon heating.

Appendix II Modified Medium for Thermus Species.

Yeast Extract 2.5 g Tryptone 2.5 g Agar 28.0 g

Na3citrate · 2H2O 0.5 mM (0.154 g)

CaSO4 · 2H2O 0.2 mM (0.04 g)

MgCl2· 6H2O 1.0 mM (0.2 g) Trace Element Solution 0.5 mL 0.01 M Fe Citrate 0.5 mL Phosphate Buffer 100.0 mL Distilled Water 900.0 mL Adjust pH to 7.2 with NaOH. Autoclave at 121 oC for 15 minutes. Autoclave the phosphate buffer separately and then add to the medium.

Phosphate Buffer:

KH2PO4 5.44 G

Na2HPO4 17.0 G Distilled Water 1.0 L Adjust the above solution to pH = 7.2.

Trace Element Solution:

Na3citrate · 2H2O 19.7 g

FeCl2· 4H2O 1.0 g

MnCl2· 4H2O 0.5 g

CoCl2· 6H2O 0.3 g

CuCl2· 2H2O 50.0 mg

Na2MoSO4 · 2H2O 50.0 mg

H3BO3 20.0 mg

NiCl2· 6H2O 20.0 mg Distilled Water 1.0 L

Notes:

1. Na3citrate · 2H2O replaces Nitrilotriacetic acid, which is a teratogen. 2. 0.01 M M Fe Citrate is made up as a 245 mg/100 ml solution in water. Fe citrate dissolves upon heating.