Comparative Bottom-Up and Middle-Down Proteomics for Escherichia-Coli
Disciplines
Analytical Chemistry | Biochemistry
Abstract (300 words maximum)
Proteomics is the large-scale analysis of proteins found in a particular cell or tissue, giving insight into the function of the cell from which the proteome originated. Proteomics involves cell lysis, in which the plasma membrane of the cell is damaged, resulting in release of all proteins from the cell. In bottom-up proteomics, cell extracted proteins are digested with trypsin generating peptides which were separated and identified by LCMS method. While bottom-up proteomics is considered the gold standard, it does introduce challenges such as peptides that are too small and some proteins are resistance to enzymatic degradation. In top-down approach, proteins are not digested, however, this makes separation and fragmentation of large proteins difficult. A new technique known as middle-down where proteins are digested to generate larger peptides by GluC enzyme or non-enzymatic chemicals. The aim of this study is to perform a comparative bottom-up and middle-down proteomics study on E. Coli using enzymatic and non-enzymatic digestions. In the bottom-up approach, trypsin/Lys C was employed which cleaves at the C-terminal of Arg/Lys whereas in middle-down approach formic acid was used which cleaves at Glu/Asp. In bottom-up approach, we have identified 3597 proteins, with the peptide mass range of 0.6-6.7 kDa. Among these proteins, 150 are related to kinase activity, 408 proteins are associated with transporter activity, 626 are shown to have nucleic acid activity, and 2182 are other proteins with unknown functions. In the middle-down approach, we have identified 1397 proteins, with the peptide mass range of 0.7- 5.65 kDa. Among these proteins, 72 proteins are associated with kinase activity, 128 have transporter activity, 242 have nucleic acid activity, and 1011 are other proteins. The top 40 proteins which are identified by both methods are not similar, which demonstrated that enzymatic and non-enzymatic digestions can help to identify diverse proteomes.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad A. Halim
Comparative Bottom-Up and Middle-Down Proteomics for Escherichia-Coli
Proteomics is the large-scale analysis of proteins found in a particular cell or tissue, giving insight into the function of the cell from which the proteome originated. Proteomics involves cell lysis, in which the plasma membrane of the cell is damaged, resulting in release of all proteins from the cell. In bottom-up proteomics, cell extracted proteins are digested with trypsin generating peptides which were separated and identified by LCMS method. While bottom-up proteomics is considered the gold standard, it does introduce challenges such as peptides that are too small and some proteins are resistance to enzymatic degradation. In top-down approach, proteins are not digested, however, this makes separation and fragmentation of large proteins difficult. A new technique known as middle-down where proteins are digested to generate larger peptides by GluC enzyme or non-enzymatic chemicals. The aim of this study is to perform a comparative bottom-up and middle-down proteomics study on E. Coli using enzymatic and non-enzymatic digestions. In the bottom-up approach, trypsin/Lys C was employed which cleaves at the C-terminal of Arg/Lys whereas in middle-down approach formic acid was used which cleaves at Glu/Asp. In bottom-up approach, we have identified 3597 proteins, with the peptide mass range of 0.6-6.7 kDa. Among these proteins, 150 are related to kinase activity, 408 proteins are associated with transporter activity, 626 are shown to have nucleic acid activity, and 2182 are other proteins with unknown functions. In the middle-down approach, we have identified 1397 proteins, with the peptide mass range of 0.7- 5.65 kDa. Among these proteins, 72 proteins are associated with kinase activity, 128 have transporter activity, 242 have nucleic acid activity, and 1011 are other proteins. The top 40 proteins which are identified by both methods are not similar, which demonstrated that enzymatic and non-enzymatic digestions can help to identify diverse proteomes.