Protein Sequencing By Edman Degradation Carried Out In A Microfluidic Chip

The sequence analysis of proteins and peptides is important in biological research, especially for those in low abundance, e.g. disease-associated proteins. Recently, mass analysis of tryptic peptides has become a powerful tool in protein identification due to the advancements in both mass Spectrometry instrumentation and bioinformatics. However, a problem of this method is its limitations in the accuracy and specificity of protein identification. Edman degradation is an accurate way to easily interpret long amino acid sequences. However, the application of Edman degradation for the identification of low abundant proteins is limited due to its low sensitivity. Here we report a novel microfluidic chip based Edman degradation system. Combined with MALDI-TOF-MS detection, the sensitivity of Edman degradation and the identification specificity of gel-separated protein in low abundance have been drastically improved.In chapter 1, the historical development and the recent advance of protein sequencing were reviewed.In chapter 2, a microfluidic based solid-phase extraction (SPE) device with capacity of 3.5 nL was developed for enrichment of gel-separated proteins in low abundance. Only 50 nL eluent were sufficient to elute effectively the truncated peptides from the nano scale column, and directly spotted onto a MALDI target without any sample loss. Analysis of low abundant proteins isolated from 2-DE with silver staining demonstrates this approach is simple and useful to improve the sequence coverage of the identified protein.In chapter 3, Edman degradation was carried out in a microfluidic device with starting peptide as little as 0.2 fmol. From the mass difference between two consecutive cycles of Edman degradation, the amino acid residue can be identified, which can be used to define the N-terminal sequence. The result demonstrated that the sensitivity of the microfluidic chip based Edman degradation system was increased by several hundred-fold compared to the current Edman degradation method. Combined with MALDI-TOF-MS detection, the identification specificity of gel-separated protein in low abundance has been drastically improved.In chapter 4, the application of the newly developed technique to de novo sequencing was investigated. Comparison between the MS/MS spectra of a native peptide and its N-terminus truncated counterpart after carrying out one cycle of Edman degradation not only can provide N-terminal residue information but also facilitates the identification of different series of fragment ions. Manual peptide sequencing is feasible and rapid by using this method as demonstrated with three peptide examples including one neuropeptide. Furthermore, two cycles of Edman degradation allows the determination of the exact value of b₂ ion of the intact peptide, which can serve as an internal calibrant to increase the mass accuracy of the MS/MS spectrum.In chapter 5, gas phase Edman degradation was carried out in a microfluidic chip combined with a sequential injection system, which was automatically manipulated by the computer. The reagent vapours were delivered by the programmed syringe pumpes. Liquid reagents in nanoliters were drawn into the microchannel by capillary force and were quantitatively transported through the reaction cartridge by the nitrogen gas in 3000 mbar. Experimental results indicate that it is possible to carry out Edman degradation automatically in fetemole level by further improving the suggested sequential injection system.