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. 2010 Nov 1;82(21):9090-9.
doi: 10.1021/ac102387t. Epub 2010 Oct 8.

Molecular detection of targeted major histocompatibility complex I-bound peptides using a probabilistic measure and nanospray MS3 on a hybrid quadrupole-linear ion trap

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Free PMC article

Molecular detection of targeted major histocompatibility complex I-bound peptides using a probabilistic measure and nanospray MS3 on a hybrid quadrupole-linear ion trap

Bruce Reinhold et al. Anal Chem. .
Free PMC article

Abstract

A nanospray MS(3) method deployed on a quadrupole linear ion trap hybrid can detect targeted peptides with high dynamic range and high sensitivity from complex mixtures without separations. The method uses a recognition algorithm that is a modification of the relative (Kullback-Leibler, KL) entropy characterization of probabilistic distance to detect if reference MS(3) fragmentation patterns are components of acquired MS(3) spectra. The recognition reflects the probabilistic structure of physical MS measurements unlike the Euclidean or inner product metrics widely used for comparing spectra. It capably handles spectra with a significant chemical ion background in contrast to the Euclidean metric or the direct relative entropy. The full nanospray MS(3) method allows both the detection and quantitation of targets without the need to obtain isotopically labeled standards. By avoiding chromatographic separations and its associated surface losses, the detection can be applied to complex samples on a very limited material scale. The methodology is illustrated by applications to the medically important problem of detecting targeted major histocompatibility complex (MHC) I associated peptides extracted from limited cell numbers.

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Figures

Figure 1
Figure 1
Probabilistic MS detection with chemical noise combines a formal calculation of a probability of target events (the M axis) plotted against an m/z translation to identify uniqueness in the M-dependent probability (m/z offset axis). The resulting probability surface is typically represented by a single contour (fixed probability) with the relative amplitude of the 0 offset peak of the contour plot related to the degree of confidence in the detection.
Figure 2
Figure 2
MS3 detection of immunodominant M158−66 influenza peptide GILGFVFTL from 107 infected and uninfected human bronchial epithelial (BEAS) cells. (A) 90 s collection of MS3 483.8:587.4 of HLA−A2 peptides isolated from infected BEAS cells produces a spectrum almost indistinguishable from the synthetic peptide (synthetic spectrum not shown). (B) Poisson detection plot for the M-dependent probability of the reference b6 (GILGFV-) fragment’s dissociation pattern in MS3 spectrum of panel A. (C) 9 min collection of MS3 483.8:587.4 from uninfected BEAS cells. (D) Poisson detection plot for the M-dependent probability of b6 fragment in MS3 spectrum C. In all four plots, the y-axis is in units of “events”; for A and C, it is recorded events; for B and D, it is M of Figure 1.
Figure 3
Figure 3
MS3 Poisson detection of influenza peptide FVANFSMEL in 10 million infected and uninfected human bronchial epithelial cells. (A) MS3 529.3:579.3 spectrum from infected sample. (B) MS3 529.3:579.3 from uninfected sample. (C) Poisson detection plot for the M-dependent probability of the reference b5 (FVANF-) fragment’s dissociation pattern in spectrum A. (D) Poisson detection plot for the probability of reference b5 pattern in spectrum B. The magnitude of the 0 m/z offset peak relative to nonzero m/z offsets is the metric of detection. As in Figure 2, the y-axis units are “events”.
Figure 4
Figure 4
Poisson detection plots for peptide GILGFVFTL in samples of 5 million T1 cells. (A, C, E) Detection of y7 fragment in the MS3 spectrum 483.79/796.46; (B, D, F) detection of b6 fragment in the MS3 spectrum 483.79:587.34. The x-axis is m/z translation of the reference pattern; the y-axis is the event number corresponding to the cutoff probability. (A, B) Five million T1 cells, the negative control. (C, D) Five million T1 cells with 829 amols or 100 copies/cell of GILGFVFTL added. (E, F) Five million T1 cells incubated in a solution of 62 pg/mL GILGFVFTL prior to cell lysis and affinity purification of HLA−A2 complexes.
Figure 5
Figure 5
MS3 483.8:796.5 spectra of control T1 cells (A) and 62 pg/mL loaded T1 cells (C) have only minor spectral differences due to the dissociation of the y7 fragment of GILGFVFTL. The overlap of major background fragments with some of the peaks expected in y7’s dissociation dominates the metric contrast and its associated sliding inner product or correlation function score (B, D). The negative sample (A, B) cannot be distinguished from the positive pair (C, D). This is in contrast to the Poisson plots of Figure 4A,E made from the same reference pattern and MS3 spectra. To improve the detection specificity of the translated inner products (B, D), the m/z range above 760 is removed (shown in gray) as the peaks in the parent window near m/z 796.5 and the associated neutral losses have little value in detection but add substantially to the 0-offset amplitude and at offsets corresponding to neutral mass increments in the correlation function plots.

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