Enhancing MALDI Time-Of-Flight Mass Spectrometer Performance through Spectrum Averaging

<div><p>Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometers are simple and robust mass spectrometers used for analysis of biologically relevant molecules in diverse fields including pathogen identification, imaging mass spectrometry, and natural products chemistry. Despite high nominal resolution and accuracy, we have observed significant variability where 30–50% of individual replicate measurements have errors in excess of 5 parts-per-million, even when using 5-point internal calibration. Increasing the number of laser shots for each spectrum did not resolve this observed variability. What is responsible for our observed variation? Using a modern MALDI-TOF/TOF instrument, we evaluated contributions to variability. Our data suggest a major component of variability is binning of the raw flight time data by the electronics and clock speed of the analog-to-digital (AD) detection system, which requires interpolation by automated peak fitting algorithms and impacts both calibration and the observed mass spectrum. Importantly, the variation observed is predominantly normal in distribution, which implies multiple components contribute to the observed variation and suggests a method to mitigate this variability through spectrum averaging. Restarting the acquisition impacts each spectrum within the electronic error of the AD detector system and defines a new calibration function. Therefore, averaging multiple independent spectra and not a larger number of laser shots leverages this inherent binning error to mitigate variability in accurate MALDI-TOF mass measurements.</p></div

Absolute values for mean, maximum and minimum errors observed for peptides from a standard protein trypsin digestion demonstrate higher variability in the more inaccurate data.

<p>Measured errors were converted to absolute values to evaluate the dispersion of data measurements and plotted according to peptide. The uneven distribution of maximum and minimum errors is expected because of zero as a lower bound for minimum error. Note, absolute value transformation of the data eliminates negative values and the mean errors reported here are higher than the mean reported for the observed peptide masses, which contain both positive and negative errors.</p

Evidence for discontinuous binning of MALDI-TOF mass spectrometry data for Des-Arg(9) Bradykinin (A), Angiotensin 1 (B), and ACTH 1–17 (C).

<p>Representative monoisotopic peaks for Des-Arg(9) Bradykinin (MH+ = 904.4676), Angiotensin 1 (MH+ = 1296.6848) and ACTH 1–17 (MH+ = 2093.0862) are enlarged to demonstrate the discontinuous sampling points (or bins) within the MALDI-TOF data. The red vertical lines are fitted to the bins evident in the observed peak shapes. The spacing of bins in mass units is larger for higher mass ions and can be accurately calculated by relationship between flight times and the ratio of masses of the molecular ions according to the equation Δt₂/Δt₁ = (M₂/M₁)^1/2. This calculation is simplistic, but accurately relates the bin spacing for different mass ions to 5 decimal places, thus suggesting that binning of data in the MALDI-TOF instrument is related to the measurements of ion flight times.</p

Effect of binning and interpolation in MALDI-TOF data.

<p>The signal detected for the flight times of a packet of ions should exhibit a continuum distribution related to the energy distribution of the population of ions (TOP). However, the AD detection system can only measure ion signal from the detector in discrete time intervals represented by the red vertical lines (TOP). The discrete intensity versus time measurements are effectively bins and produce the observed discontinuous peak profile (BOTTOM). This discontinuous data must be interpolated by peak fitting algorithms to estimate the parameters of the original continuum spectrum and derive accurate mass measurements.</p

Beta-Tubulin identification and limitations of peptide mass fingerprinting.

<p>The identification of tubulin from peptide mass fingerprinting matches with LC-MS/MS data for the majority of peptides. However, assignment for the 1249.585 and 1696.805 peptides were inaccurate. The 1249.585 peak was not assigned in the MALDI-TOF data. The observed mass of 1696.805 and data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120932#pone.0120932.t003" target="_blank">Table 3</a> are from ProFound and assigned to peptide sequence ALTVSELTQQMFDSK (data denoted with an asterisk *). Peptide fragmentation data using LC-MS/MS suggests this assignment is incorrect and that the correct sequence is NSSYFVEWIPNNVK with deamidation at the amino-terminus yielding the sequence DSSYFVEWIPNNVK (MH+ = 1697.817 expected, 1697.813 observed, −2.4 ppm error).</p><p>Beta-Tubulin identification and limitations of peptide mass fingerprinting.</p

Identification of tubulin peptides by ProFound peptide mass fingerprinting using immunoprecipitation experiments and averaged MALDI-TOF/TOF data.

<p>Tubulin was immunoprecipitated from HEK293 cell lysate using a rabbit polyclonal antibody and Protein A/G Dynabeads. After isolation, all eluted proteins were reduced and alkylated, digested with trypsin, and analyzed on an ABI 4800 MALDI-TOF/TOF mass spectrometer. Multiple individual spectra were acquired with internal calibration and between 10 and 23 individual measurements for each peptide were used for calculating the average observed masses for each peptide. Mass Tolerance is in parts-per-million (ppm), Peptide Set defines the peptides included for search and Search Mass Range/pI Range are input parameters for Profound. Top Protein ID and Expectation Value were calculated within ProFound from the mass spectrometry data using the IPI Human database (2010-02-01).</p><p>Identification of tubulin peptides by ProFound peptide mass fingerprinting using immunoprecipitation experiments and averaged MALDI-TOF/TOF data.</p

Serum Human C-peptide levels in nu/nu mice transplanted with human fetal ICCs treated with AMD3100.

<p>Sixteen weeks after transplantation, circulating human C-peptide levels were measured 30 minutes after a glucose challenge in fasted nu/nu mice that had been treated with saline or AMD3100. For both the saline and AMD3100 groups n = 5. For the control group, C-peptide levels were 517.3±198.83 (mean±SEM). C-peptide levels in the AMD3100 treatment group were undetectable. P<0.05 by Student’s t-test.</p

SDF-1α stimulates Akt but not MAPK phosphorylation in CFPAC-1 cells and fetal ICCs.

<p>Following 48 hr serum starvation, CFPAC-1 cells were stimulated with 100 ng/ml or 300 ng/ml human recombinant SDF-1α for 10 min at 37°C. Whole cell lystes were analyzed by western blot, using antibodies raised against dually phosphorylated phospho-MAPK(ERK1/ERK2)(A) or phospho-Akt (Ser473)(B). Following overnight serum starvation, ICCs from human fetal pancreas of 15 weeks gestation were stimulated with 100 ng/ml or 300 ng/ml SDF-1α for 10 min at 37°C. Whole cell lystes were analyzed by western blot, using an antibody raised against phospho-MAPK(C) or phospho-Akt (Ser473) (D). All blots were stripped and reblotted with antibodies to total Akt, Erk, and Hsp90 sequentially to confirm equal loading.</p

The effect of the inhibitors of PI 3-kinase, MAPK, PLC, and PKA on SDF-1α stimulated proliferation in CFPAC-1 cells.

<p>CFPAC-1 cells were seeded in 12 well plates and grown to 50–70% confluence. Following 24 hr serum starvation, cells were pre-treated with LY294002 (30 µM), U0126 (30 µM), Edelfosine (10 µM), or H89 (10 µM) for 30 minutes and stimulated with SDF-1α for 16 hrs. BrdU was added 4 hrs before fixation. BrdU incorporation was determined as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038721#s4" target="_blank">Materials and Methods</a>. (*, P<0.05 SDF-1 versus basal); (**, p<0.02 SDF-1/Edelfosine versus SDF-1 alone); (***, p<0.0001 SDF-1/LY294002 and SDF-1/U0216 versus SDF-1 alone) by Student’s t-test. N.S.  =  not significant by Student’s t-test.</p

The cytokine cocktail TNFα, IL1β and IFNγ and SDF-1α (300 ng/ml) stimulate CFPAC-1 apoptosis.

<p>CFPAC-1 cells were seeded in 12 well plates and grown to 50–70% confluence. Following 24 hr serum starvation, cells were stimulated with either the cytokine cocktail (TNF) consisting of IL-1β (2 ng/ml), IFN-γ (100 ng/ml) and TNF-α (100 ng/ml), or SDF-1α at 100 ng/ml, 300 ng/ml, or the TNF cocktail in combination with SDF-1α 100/ng/ml for 24 hrs. The cells were fixed at the end of the incubation apoptosis was quantitated using the TUNEL method as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038721#s4" target="_blank">Materials and Methods</a>. The number of TUNEL positive nuclei was expressed as the percentage of the total number cells counted in the acquired images. Data is expressed as mean ± SEM. (*, p<0.03 versus basal); (**, p<0.01 versus basal) by Student’s t-test. N.S.  =  not significant by Student’s t-test.</p