Online nanoflow reversed phase-strong anion exchange-reversed phase liquid chromatography-tandem mass spectrometry platform for efficient and in-depth proteome sequence analysis of complex organisms

Abstract

The dynamic range of protein expression in complex organisms coupled with the stochastic nature of discovery-driven tandem mass spectrometry (MS/MS) analysis continues to impede comprehensive sequence analysis and often provides only limited information for low-abundance proteins. High-performance fractionation of proteins or peptides prior to mass spectrometry analysis can mitigate these effects, though achieving an optimal combination of automation, reproducibility, separation peak capacity, and sample yield remains a significant challenge. Here we demonstrate an automated nanoflow 3-D liquid chromatography (LC)-MS/MS platform based on high-pH reversed phase (RP), strong anion exchange (SAX), and low-pH reversed phase (RP) separation stages for analysis of complex proteomes. We observed that RP-SAX-RP outperformed RP-RP for analysis of tryptic peptides derived from Escherichia coli and enabled identification of proteins present at a level of 50 copies per cell in Saccharomyces cerevisiae, corresponding to an estimated detection limit of 500 amol, from 40 μg of total lysate on a low-resolution 3-D ion trap mass spectrometer. A similar study performed on a LTQ-Orbitrap yielded over 4000 unique proteins from 5 μg of total yeast lysate analyzed in a single, 101 fraction RP-SAX-RP LC-MS/MS acquisition, providing an estimated detection limit of 65 amol for proteins expressed at 50 copies per cell.

Figure 1

Schematic diagram of automated, online nanoflow RP-SAX-RP platform. (A) The autosampler first loads sample and then is used to inject first- (acetonitrile, orange) or second- (KCl, blue) dimension eluents, respectively. (B, C) An additional 6-port, 2-position valve provides a vented third dimension column configuration and ensures efficient capture of peptides on the low-pH RP precolumn. (B) Second dimension fractions are diluted (4:1) and acidified with reversed phase solvent A (0.1% formic acid, 3% acetonitrile) introduced by an ultra-high pressure binary pump. (C) An organic gradient is delivered by the binary pump to elute peptides from the third dimension column for MS/MS analysis. The vent valve generates a pre-column effluent split of ≈1000:1 to provide a stable column/ESI flow rate of ≈5-10 nL/min. Active solvent flow paths are highlighted in green. (D) A computer-controlled positioning platform (Digital PicoView) automatically moves the emitter tip between “electrospray” and “wash” positions during data acquisition and sample loading, respectively.

Figure 2

(A) Replicate analyses of tryptic peptides derived from e.coli lysate by RP-RP (left, red) and RP-SAX-RP (right, blue) fractionation on a LCQ Deca mass spectrometer. RP-SAX-RP provided for higher numbers of identified peptides and proteins as compared to RP-RP for independent experiments spanning 37 and 40 fractions, respectively. (B) Peptide elution profiles, defined as the number of first (high-pH RP) or first and second (high-pH RP and SAX), fractions spanned by peptides detected in (A). (C) Distribution of unique peptide identifications in a 37 fraction RP-SAX-RP experiment.

Figure 3

Technical replicate 19 fraction RP-SAX-RP analyses of s.cerevisiae tryptic peptides on a LTQ-Orbitrap mass spectrometer. (A) Venn diagrams illustrate the reproducibility of peptide (≈55%) and protein (≈75%) identifications. (B) Histogram distribution of Log₂ ratios for peptide precursors matched by unique amino acid sequence or a combination of intact peptide mass (±10 ppm) and third dimension LC elution time (± 0.5 min).

Figure 4

(A, B) Proteome coverage for s.cerevisiae as a function fractionation depth (19, 51, 101, 236 fractions) and mass spectrometry platform (LCQ Deca or LTQ-Orbitrap XL) as compared to biochemical-based quantification of protein expression. Western blot data (orange) are used as a reference set within each expression level bin. The count of reference proteins detected in each RP-SAX-RP experiment is plotted with the color scheme as indicated in the legend. The x-axis is labeled based on absolute expression level (copies per cell as determined from TAP-GFP data) or relative protein quantity calculated from absolute expression level and total lysate analyzed by each RP-SAX-RP experiment (LCQ – 40 μg, LTQ-Orbitrap – 5 μg). (C) Expression histograms plotted as in (A, B) for protein groups from Ghaemmaghami, et al, “Extremely low signal (< 50 copies/cell)” and “No expression detected,” not amenable to absolute quantification by western blot.

Figure 5

Expression histogram as in Figure 4 (A), plotted as a percentage of the total protein count detected in each experiment. TAP-GFP data (orange) serves as the reference protein set in each expression bin. The x-axis is labeled based on absolute expression level (copies per cell as determined from TAP-GFP data) or relative protein quantity calculated from absolute expression level and total lysate analyzed by each RP-SAX-RP experiment (LCQ – 40 μg, LTQ-Orbitrap – 5 μg).