Production and analysis of multiply charged negative ions by liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

Abstract

Rationale: Liquid atmospheric pressure matrix-assisted laser desorption/ionisation (AP-MALDI) has been shown to enable the production of electrospray ionisation (ESI)-like multiply charged analyte ions with little sample consumption and long-lasting, robust ion yield for sensitive analysis by mass spectrometry (MS). Previous reports have focused on positive ion production. Here, we report an initial optimisation of liquid AP-MALDI for ESI-like negative ion production and its application to the analysis of peptides/proteins, DNA and lipids.

Methods: The instrumentation employed for this study is identical to that of earlier liquid AP-MALDI MS studies for positive analyte ion production with a simple non-commercial AP ion source that is attached to a Waters Synapt G2-Si mass spectrometer and incorporates a heated ion transfer tube. The preparation of liquid MALDI matrices is similar to positive ion mode analysis but has been adjusted for negative ion mode by changing the chromophore to 3-aminoquinoline and 9-aminoacridine for further improvements.

Results: For DNA, liquid AP-MALDI MS analysis benefited from switching to 9-aminoacridine-based MALDI samples and the negative ion mode, increasing the number of charges by up to a factor of 2 and the analyte ion signal intensities by more than 10-fold compared with the positive ion mode. The limit of detection was recorded at around 10 fmol for ATGCAT. For lipids, negative ion mode analysis provided a fully orthogonal set of detected lipids.

Conclusions: Negative ion mode is a sensitive alternative to positive ion mode in liquid AP-MALDI MS analysis. In particular, the analysis of lipids and DNA benefited from the complementarity of the detected lipid species and the vastly greater DNA ion signal intensities in negative ion mode.

FIGURE 1

Liquid AP‐MALDI‐Q‐TOF MS spectra of ubiquitin analysed using three different matrix chromophores in positive A‐C, and negative D‐F, ion modes: DHB A‐D, CHCA B‐E, and 9‐AA C‐F. The data was acquired over 1 min at a laser pulse repetition rate of 10 Hz. The bin size was set to m/z 1 with minimum background subtraction. The most abundant charge state is marked by an asterisk

FIGURE 2

Centroided liquid AP‐MALDI‐QTOF MS spectra using DHB‐based liquid MALDI samples in positive ion mode A‐C, and 9‐AA‐based liquid MALDI samples in negative ion mode D‐F: DNA1 A‐D, DNA2 B‐E, and DNA3 C‐F. The data was acquired over 1 min at a laser pulse repetition rate of 10 Hz. For b, c, e and f, the ion signal intensity is magnified by a factor of 5 for the ions above m/z 750 as indicated in the top of the spectra. Insets show zoom‐ins for some of the multiply charged analyte ions

FIGURE 3

Unprocessed liquid AP‐MALDI‐Q‐TOF MS spectra indicating the limit of detection for DNA1 [M − 2H]²⁻ (m/z 894.19). The absolute amount per sample droplet can be found in the top left corner of the spectra. The data was acquired over 1 min at a laser pulse repetition rate of 10 Hz. In all cases only a fraction of the sample was consumed during the 1‐min acquisition. Italic m/z values mark ion signals not related to the analyte

FIGURE 4

Liquid AP‐MALDI‐Q‐TOF MS spectra of lipid extracts from bovine whole milk analysed in positive A, B, and negative C, D, ion mode using DHB A, C, and 9‐AA B, D, as MALDI matrix chromophore. The ion signal intensity is magnified for the ions above m/z 500 as indicated in the top of the spectra. Phospholipid [M + H]⁺ and [M − H]⁻ ions putatively identified from these spectra can be found in Table S1 (supporting information)