Development and Applications of Stable Isotope Labelling Liquid Chromatography Mass Spectrometry for Quantitative Proteomics

• Author / Creator

  Lo, Andy

• This thesis describes the characterization, automation, and applications of a stable isotope labelling method for quantitative proteomics by mass spectrometry. Known as 2MEGA, the method uses guanidinylation to convert peptide lysine residues to homoarginine followed by reductive methylation of free peptide N-termini with isotopically encoded formaldehyde. 2MEGA was shown to be applicable to the identification of membrane proteins by increasing the percentage of lysine-containing peptides identified and by the observation of diagnostic a1-ions for >95% of glycine N-terminal peptides and >99% of non-glycine N-terminated peptides. Subsequent work demonstrated that 2MEGA was readily automatable with a commercially available liquid handler. With minor reagent substitutions, the 2MEGA labelling method was used to simultaneously process twelve samples. Over 98% labelling efficiency was observed, with the most common side reaction products being N-terminal guanidinylation (~2%) for glycine and alanine N-terminal peptides. Various front-end protein sample preparation methods were found to be compatible with the procedure. Reciprocal labelling was used to evaluate the internal consistency from quantitative peptide sample comparisons by switching the original isotopic labelling assignment and analyzing the resultant sample mixture. With approximately 60% overlap in peptide identifications, reversal of the isotopic labels was not found significantly affect the observed quantification ratios, as evidenced by the internal consistency in the peptide quantification ratios (an average of 1.29-fold relative difference for the entire E. coli dataset). For over 90% of peptides, the relative error was less than 50%. After discarding 1% of the quantified peptides, approximately 1% of protein matches were lost, but with significant gains in the internal consistency of quantification values. The large-scale quantitative analysis suggested that data processing can greatly influence the overall consistency observed in proteomics experiments. Reciprocal labelling was also applied to the analysis of a human carcinoma cell line deficient in Bax, a key protein in stimuli-induced apoptosis, in which 200 proteins with a significant change abundance difference were identified from Bax-expressing and Bax-deficient samples. Overall, the thesis highlights the potential of the 2MEGA labelling method, its applicability to high throughput MS-based proteomics applications, and suggests an increased role for quantitative mass spectrometry as a routine bioanalytical methodology.

• Subjects / Keywords

  • Proteomics
  • Isotope labeling
  • Quantitation
  • Mass-spectrometry

• Graduation date

  Spring 2011

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3332K

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.