Single-molecule mass spectrometry in solution using a solitary nanopore
- Joseph W. F. Robertson*,
- Claudio G. Rodrigues†,
- Vincent M. Stanford‡,
- Kenneth A. Rubinson*,§,
- Oleg V. Krasilnikov†,¶, and
- John J. Kasianowicz*,¶
- *Electronics and Electrical Engineering Laboratory, Semiconductor Electronics Division, National Institute of Standards and Technology, Gaithersburg, MD 20899-8120;
- †Laboratory of Membrane Biophysics, Department of Biophysics and Radiobiology, Universidade Federal de Pernambuco, 50670-901, Recife, PE, Brazil;
- ‡Information Technology Laboratory, Information Access Division, National Institute of Standards and Technology, Gaithersburg, MD 20899-8940; and
- §Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435
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Edited by Nicholas J. Turro, Columbia University, New York, NY, and approved March 29, 2007 (received for review December 13, 2006)
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Fig. 1.Neutral polymers cause well defined reductions in the ionic current as they partition into a solitary nanopore in a lipid bilayer membrane. (Middle Left) The ionic current, through an αHL channel bathed by a polymer-free solution, is quiescent. Addition of polydisperse PEG (M r = 1,500 g/mol) (Top Right) cause persistent current blockades (Middle Right and Bottom). The solutions bathing the membrane contained 4 M KCl and 5 mM Tris buffer, pH 7.5. (Middle and Bottom) The horizontal dashed lines indicate zero current.
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Fig. 2.A single nanopore discriminates between polymers with different molecular masses. The difference between the conductance states caused by polydisperse (M r = 1,500) (Lower Left) and monodisperse (M = 1,294 g/mol, n = 29) (Upper Left) PEG is readily apparent. The time series data shown contained ∼500 and ∼700 events for the poly- and monodisperse PEG samples, respectively. (Upper and Lower Right) All-points histograms of the ionic current reflect the distinct natures of the two polymer samples. The ionic current histograms for each sample were calculated from >105 blockade events. The long-lived, small ionic current blockades near zero in the monodisperse PEG time series are most likely caused by impurities in the PEG samples. These events are long-lived but few in number.
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Fig. 3.Mass distributions obtained with a single nanopore (Upper) is compared with a conventional MALDI-TOF mass spectrum (Lower) for polydisperse PEG (M r = 1,500 g/mol). The histogram was obtained as described in the text. Greater values of I/I open correspond to lower PEG molecular masses. The histogram of the state-averaged current (red) are overlaid with the GMM fit (black). The model fits the empirical probability density function well with a Kolmogorov–Smirnov goodness of fit statistic, KS = 0.295 (32). The mean conductance-based histogram for monodisperse PEG-1294 (blue) is scaled to the height of the corresponding polydisperse peak. In the MALDI-MS, under the desorption/ionization conditions used, each PEG n-mer yields a parent ion peak, MH+, and a base peak 16–17 units lower in mass, suggesting a loss of −O or −OH.
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Fig. 4.The current through a solitary nanopore discriminates between individual PEG polymers that have different molecular masses. Ionic current blockades caused by individual molecules are assigned to Gaussian states of the nanopore mass spectrogram (Fig. 3 Upper). The GMM permits assignment of individual blockades to the conductance states by maximum likelihood decoding (solid black lines). (Upper) A 15-second-long block of data showing the open channel and blockade states. Expansion of the time series data in the highlighted region (Lower Left) compared with a histogram made from the GMM fit (Lower Right). The colored peaks in the histogram reference the individual polymers in the pPEG that are discussed in Fig. 5.
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Fig. 5.Residence-time distributions associated with each polymer species vary systematically with the polymer mass. The derived residence time distributions are shown on a semilog plot for three representative states corresponding to the 1,294 (red), 1,558 (green), and 2,042 (blue) g/mol components of pPEG and to mPEG 1294 (black). The mean residence times, estimated from a least-squares fit of a single exponential to each data set are (in milliseconds) as follows: (2.8 ± 0.1), (3.2 ± 0.1), (13.4 ± 0.1), (52 ± 2) for mPEG 1294, pPEG 1294, pPEG 1558, and pPEG 2042, respectively.
Footnotes
- ¶To whom correspondence may be addressed. E-mail: kras{at}ufpe.br or john.kasianowicz{at}nist.gov
- © 2007 by The National Academy of Sciences of the USA
