Ultrasensitive proteomics using high-efficiency on-line micro-SPE-NanoLC-NanoESI MS and MS/MS

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of similar to10(3)) 15-mum-i.d. capillary liquid chromatography separations (i.e., using nanoLC, similar to20 nL/min mobile-phase flow rate at the optimal linear velocity of similar to0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/ FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., similar to100 times larger than FTICR MS), corresponding to a sensitivity of similar to7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.