(A). BPI chromatograms of the online digestion of 15 pmoles of phosphorylase B and separation at 20–21 °C (see Experimental section for exact gradient information). Six independent experiments have been overlaid. The region of the chromatogram (elution times 3.75–5.75 minutes) with the highest density of peaks is shown. (B). Separation as a function of temperature. BPI chromatograms of 15 pmoles phosphorylase B digested online and separated at the following chamber temperatures: 21 °C, 10°C, 5°C, 1°C. The (*) and (◊) indicate the positions at which the example mass spectra in panel (C) were obtained. The bar above the top trace indicates the region of the data shown in panel A. (C). Cumulative mass spectra between elution times 4.2 and 4.4 minutes. The top spectrum is the sum of 12 scans taken between elution times 4.2 and 4.3 minutes (* in panel B) and the bottom spectrum is the sum of 12 scans taken between elution times of 4.3 and 4.4 minutes (◊ in panel B). Data are magnified 10x for m/z range 700–1000 and 550–1000 for the top and bottom spectra, respectively. (D). Demonstration of the improvement in separation at 0 °C with different chromatographic media. In each case, the same 52 kDa protein was digested with pepsin and separated. BPI chromatograms of digested protein are shown. (i). 200 pmoles of digested protein desalted using a POROS 20-R2 trap and separated using a 0.5 × 100 mm perfusion column packed with POROS 20-R2 (20 µm particle size), 2–55% ACN in 9 mins. (ii). 90 pmoles of digested protein desalted on a 1 mm × 8 mm C-18 peptide trap and separated using a Zorbax C-18, 3.5 µm 300 Å, 1.0 mm × 50 mm column, 8–40% ACN in 6 mins. (iii). 10 pmoles of digested protein desalted using a 1 mm × 5 mm, ACQUITY UPLC BEH C18, 1.7 µm pre-column and separated using an ACQUITY UPLC BEH C18 1.7 µm 1.0 × 100 mm column, 8–40% ACN in 11 mins. (E). BPI chromatogram obtained during separation of a mixture of proteins totaling 249.8 kDa. The proteins were: 20 pmoles yeast enolase, 20 pmoles yeast alcohol dehydrogenase, 30 pmoles bovine serum albumin, and 60 pmoles rabbit phosphorylase B. This separation was performed at an average chamber temperature of −0.5°C.

(A). Photograph of the modified ACQUITY system. The pumps, cooling module and control electronics are illustrated: CM-cooling module; EM-electronics module; BSM-binary solvent manager; ASM-auxiliary solvent manager. (B). Schematic of the cooling module from the front. Key components: 1-pepsin column housing; 2-injection port; 3-heat sinks; 4-injection valve; 5-switching valve; 6-0.25” aluminum plate for thermal mass; 7-insulation; 8-Peltier unit. The flow path is the same as has been published previously9. (C). Top-down view of the cooling module. Heat generated by the Peltiers is displaced via circulating coolant at the back of the unit. The cooled chamber is in the front of the unit. The key components are as in panel B.

(A). BPI chromatogram of the UPLC separation of cytochrome c peptides at 0 °C. The chromatographic peaks representing four peptides used to validate the instrument are indicated on the chromatogram. 1: peptide 1–10; 2: peptide 37–46; 3: peptide 67–82; and 4: peptide 95–104. (B–E). Deuterium incorporation with time (log scale) for four peptic peptides of cytochrome c acquired using the HPLC system () or the UPLC module (). The final data point on each plot is for the heavily deuterated form (HD) of each peptide (see Experimental section). The error bars represent the worst error seen for these data points, ± 0.25 Da over the course of three replicates.