Structural characterization of phospholipids and peptides directly from tissue sections by MALDI traveling-wave ion mobility-mass spectrometry

Abstract

Ion mobility-mass spectrometry (IM-MS) provides rapid two-dimensional separations based on analyte apparent surface area or collision cross section (CCS, A(2)) and mass-to-charge, respectively. Recently, traveling-wave (t-wave) IM-MS was developed which uses electrodynamic rather than electrostatic fields commonly used in drift cell IM-MS instruments. The underlying theory for obtaining CCS data is well developed for drift cell IM-MS, while strategies for obtaining CCS values from t-wave IM-MS data remains an active area of research. In this report, methods were developed and validated to obtain CCS values of phospholipids and peptides directly from thin tissue sections by MALDI t-wave IM-MS using CCS calibrants measured by MALDI drift cell IM-MS. Importantly, the average percent difference between t-wave and drift cell CCS measurements is minimized by calibrating with the same biomolecular class. Calibrating t-wave phospholipid CCS values with drift cell peptide CCS measurements results in an average percent difference of ca. 7% between the same lipids measured using t-wave and drift cell IM-MS, while this improves to <0.5% when drift cell phospholipid CCS values are used for calibrating t-wave data. A suite of CCS values are reported for lipids and peptides that were determined directly from tissue, i.e. without the need for tissue extraction and further purification steps.

FIGURE 1

Measuring CCS values of phospholipids and tryptic peptides directly from rat brain tissue sections by MALDI t-wave IM-MS. A typical two-dimensional t_d versus m/z plot of a rat brain tissue section after trypsin application collected by MALDI t-wave IM-MS. The predicted correlation of drift time versus m/z for phospholipids and tryptic peptides at these experimental conditions are indicated by dashed lines for visualization purposes. Signal intensity is indicated by the displayed false color scale.

FIGURE 2

Calibration curves of normalized CCS versus effective drift time used to estimate CCS values of (A) phospholipids and (B) tryptic peptides directly from tissue sections. Data from 5 different wave heights are each plotted whereby the correlation line represents a power fit to the data. Since t-wave IM-MS CCS values were calculated using the t_d values obtained directly from a complex sample, only t_d values for peaks containing a single species when analyzed by FTICR were used. Calibration curves of phospholipid standards have R² values > 0.95 and calibration curves of BSA tryptic peptides have R² values > 0.98.

FIGURE 3

Comparison of PC CCS results based on using tryptic peptide CCS and phospholipids CCS values as calibrants. MALDI t-wave IM-MS CCS values for PC species that were calibrated using either tryptic peptide CCS values (red) or phospholipid CCS values (blue) measured using MALDI drift cell IM-MS or CCS values (green) measured by ESI drift cell IM-MS., , Error bars represent ± 1σ for 5 different wave heights collected over 2 days (n=10).

FIGURE 4

MALDI t-wave IM-MS CCS values of phospholipids obtained directly from tissue sections. Specific phospholipid species were identified by FTICR and MS/MS directly from tissue sections. MALDI t-wave IM-MS CCS values were determined using drift cell IM-MS phospholipid CCS values as calibrants. Error bars represent ± 1σ for 5 different wave heights collected over 2 days (n=10). The two SM species which fall on the PC trend-line were identified as [SM 22:0 + Na]⁺ and [SM 22:0 + K]⁺.

FIGURE 5

MALDI t-wave IM-MS tryptic peptide CCS values from an in-solution digest. T-wave IM-MS CCS values of tryptic peptides from a BSA digest were determined using BSA tryptic peptide CCS values measured by MALDI drift cell IM-MS as calibrants. The error bars (all are < 2.0%) represent ± 1σ for 5 different wave heights collected over 2 days (n=10). Correlation between MALDI drift cell IM-MS and MALDI t-wave IM-MS CCS values result in an R² value > 0.98, where the dashed line represents a 1:1 correlation.

FIGURE 6

MALDI t-wave IM-MS CCS values of tryptic peptides measured directly from tissue sections. CCS values were calibrated using MALDI drift tube IM-MS CCS values of BSA tryptic peptides as calibrants. Only the CCS values of confidently identified tryptic peptides sequenced by high mass accuracy and MS/MS directly from tissue sections are displayed (Table 3). Error bars (average error is < 1.5%) represent ± 1σ for 5 different wave heights collected over 2 days (n=10). For reference, SM and PC trend-lines are plotted illustrating that peptides have a lower CCS than phospholipid species of the same m/z.

Figure 7

Validation of MALDI t-wave IM-MS CCS values directly from tissue in comparison with those from an in solution digest. Both MALDI t-wave IM-MS and MALDI drift cell IM-MS CCS values are an average of 5 different wave heights and electrostatic fields, respectively, over two days (n = 10). Error bars represent ± 1σ. The dashed line represents a 1:1 correlation between MALDI t-wave IM-MS and MALDI drift cell IM-MS CCS values.