One-hour proteome analysis in yeast

Abstract

Recent advances in chromatography and mass spectrometry (MS) have made rapid and deep proteomic profiling possible. To maximize the performance of the recently produced Orbitrap hybrid mass spectrometer, we have developed a protocol that combines improved sample preparation (including optimized cellular lysis by extensive bead beating) and chromatographic conditions (specifically, 30-cm capillary columns packed with 1.7-μm bridged ethylene hybrid material) and the manufacture of a column heater (to accommodate flow rates of 350-375 nl/min) that increases the number of proteins identified across a single liquid chromatography-tandem MS (LC-MS/MS) separation, thereby reducing the need for extensive sample fractionation. This strategy allowed the identification of up to 4,002 proteins (at a 1% false discovery rate (FDR)) in yeast (Saccharomyces cerevisiae strain BY4741) over 70 min of LC-MS/MS analysis. Quintuplicate analysis of technical replicates reveals 83% overlap at the protein level, thus demonstrating the reproducibility of this procedure. This protocol, which includes cell lysis, overnight tryptic digestion, sample analysis and database searching, takes ∼24 h to complete. Aspects of this protocol, including chromatographic separation and instrument parameters, can be adapted for the optimal analysis of other organisms.

Figure 1|

Structure of the in-house-manufactured column heater. Labeled rendering shows the column oven without a lid. (1) Holes for 4–40 ×¾-inch screws to attach the four layers to one another. (2) Polycarbonate micro-tee retainer. (3) Holes to accept pins from lids (not shown). (4) Hole for resistance thermometer. (5) Hole for grounding wire. (6) Slot for foil heater connections. (7) Spring-loaded column tip retainer. (8) Undercut top layer. (9) Layer containing thermometer, heater (against bottom), and grounding wire. (10) Aluminum layer between heater and polycarbonate base plate. (11) Polycarbonate base plate. (12) Polycarbonate attachment to the nanospray ion (NSI) source.

Figure 2|

Column fabrication. (a) To pull an emitter tip, first cut an appropriate length (35–40 cm) of 360 pm o.d. × 75 μm i.d. fused silica. Remove ~2 cm of polyimide coating 3–4 cm from one end of the fused silica. (b) The column is packed with 1.7-μm BEH C18 packing material according to the protocol. (c,d) Setup of the pressure bomb.

Figure 3|

Effect of MS¹ AGC target, resolution and MS² max inject time on the number of MS/MS scans, PSMs and unique peptides.

Figure 4|

Yeast peptide and protein identifications for all replicates. (a) Plots the number of cumulative unique yeast peptide identifications for five technical replicates across the LC-MS/MS gradient. (b) Plots the corresponding proteins across the gradient. Cumulative peptide and protein identifications were determined using the Coon OMSSA Proteomic Analysis Software Suite. PSMs passing a 1% FDR cutoff were exported to a text file and processed by a modified version of Protein Hoarder (version 2.4.1). These PSMs were iteratively processed in successive 1-min windows and grouped into proteins using the law of parsimony at a 1% FDR.

Figure 5|

Unique peptides and proteins identified over the LC-MS/MS gradient. (a) Plots the number of unique yeast peptides identified in 5-min bins for five technical replicates across the LC-MS/MS gradient. (b) Plots the corresponding proteins across the gradient.

Figure 6|

Effect of gradient length on peptide and protein identifications. (a) Plots the number of cumulative unique yeast identifications for LC-MS/MS gradients of 30, 45, 70, 120, 180 and 240 min. (b) Plots the corresponding protein identifications for each gradient.