Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: proteomics on the fly

Abstract

A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure cycling technology in the range of 5-35 kpsi was demonstrated for proteomic analysis. Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 s and then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance in terms of the number of Shewanella oneidensis peptides and proteins identified in a shotgun approach was evaluated relative to a traditional "overnight" sample preparation method. Advantages of the new method include greatly simplified sample processing, easy implementation, no cross contamination among samples, and cost effectiveness.

Figure 1

a) LC-MS/MS chromatograms of high pressure assisted digestions as a function of digestion pressure. High pressure in-solution digestions of BSA were performed at 5, 10, 20, and 35 kpsi . b) Histograms depict the number of unique identified peptides for each method.

Figure 2

a) Unique peptides identified as a function of the number of PCT cycles performed at 35 kpsi and with different organic solvent compositions. Ammonium bicarbonate buffer digestions correspond to the diagonal lines; 20% methanol (v/v) digestions, to the vertical lines; and 80% methanol (v/v) digestions, to the solid white bars. b) Chromatograms corresponding to the LC-MS/MS of PCT assisted digestion performance as a function of the number of cycles and the organic content in the digestion buffer. 4- and 8-cycle high pressure in-solution digestions of BSA were performed, using ammonium bicarbonate buffer, 20% methanol, or 80% methanol.

Figure 3

Comparison of a traditional digestion protocol versus a PCT- assisted digestion. Panels a) and b) show chromatograms obtained from the LC-MS/MS analysis from traditional and PCT digestions, respectively. Panel c) compares the number of identified peptides in both samples at a given FDR following SEQUEST analysis. Filled circles correspond to the regular digestion protocol and empty circles to the PCT-assisted digestion protocol. d) Histogram showing the number of identified peptides with missed cleavages. Grey bars correspond to the traditional digestion protocol and black bars to the PCT-assisted digestion protocol. e) Overlap between the identified proteins. PCT-assisted (I) digestion protocol and the traditional (II) digestion protocol. f) Comparison of the different identified peptides in terms of trypsin specificity.

Figure 4

ESI mass spectra of horse myoglobin dissolved in 12.5 mM ammonium bicarbonate. The upper panel shows the pressure treated protein yielding denaturated horse myoglobin and elimination of the heme group. Bottom panel shows the native myoglobin without pressure treatment and the heme group attached.