Ultrasensitive Proteomics using High-Efficiency on-Line Micro-SPE-NanoLC-NanoESI MS and MS/MS
New approaches for ultra-sensitive proteomics are described for the characterization of complex protein (proteomic) samples of&< 50 ng total mass. Ultra-high sensitivity was achieved using high-efficiency 15-m i.d. capillary liquid chromatography (i.e. nanoLC) coupled on-line to a high-sensitivity Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS) through a nanoscale electrospray ionization (nanoESI) interface. The high separation efficiency (peak capacities of {approx}103 with average peak widths of {approx}15 s) and small mobile phase flow rates ({approx}20 nL/min at optimal linear velocities of {approx}0.2 cm/s) from the nanoLC and the resulting high ionization efficiency of the nanoESI provided confident protein identification from < 75-zeptomole of individual proteins (e.g. with 6 tryptic peptides from albumin) and an estimated {approx}10 zeptomole ({approx}6000 molecules) sensitivity for peptide detection. Application of the nanoLC with ion trap MS/MS also allowed targeted protein identification at low attomole levels. The on-line coupled micro solid phase extraction allowed loading of sample solutions at 8 L/min, and provided a 250 attomolar peptide concentration detection limit using FTICR MS. This sensitivity enabled identification of proteins from 0.5 pg of a whole proteome extract tryptic digest sample. The proteome measurement dynamic range, protein identification overlap, and proteome quantitation accuracy were also investigated. An modified accurate mass and time tag data analysis methodology was used for peptide and protein identification, allowing the nanoLC-FTICR MS approach to identify 872 proteins from a 3 hour analysis of a 2.5 ng Deinococcus radiodurans proteome sample. The zeptomole level sensitivity provides a basis for extension of proteomics studies to low numbers of cells, and potentially a single mammalian cell.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC06-76RL01830
- OSTI ID:
- 15007559
- Report Number(s):
- PNNL-SA-38244; KP1102010
- Journal Information:
- Analytical Chemistry, 76(1):144-154, Journal Name: Analytical Chemistry, 76(1):144-154
- Country of Publication:
- United States
- Language:
- English
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