Applying mass spectrometry to study non-covalent biomolecule complexes.

Non-covalent interactions are essential for the structural organization of biomacromolecules and play an important role in molecular recognition processes, such as the interactions between proteins, glycans, lipids, DNA, and RNA. Mass spectrometry (MS) is a powerful tool for studying of non-covalent interactions, due to the low sample consumption, high sensitivity, and label-free nature. Nowadays, native-ESI MS is heavily used in studies of non-covalent interactions and to understand the architecture of biomolecular complexes. However, MALDI-MS is also becoming increasingly useful. It is challenging to detect the intact complex without fragmentation when analyzing non-covalent interactions with MALDI-MS. There are two methodological approaches to do so. In the first approach, different experimental and instrumental parameters are fine-tuned in order to find conditions under which the complex is stable, such as applying non-acidic matrices and collecting first-shot spectra. In the second approach, the interacting species are "artificially" stabilized by chemical crosslinking. Both approaches are capable of studying non-covalently bound biomolecules even in quite challenging systems, such as membrane protein complexes. Herein, we review and compare native-ESI and MALDI MS for the study of non-covalent interactions.

A new, modular mass calibrant for high-mass MALDI-MS.

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of high-mass proteins requires suitable calibration standards at high m/z ratios. Several possible candidates were investigated, and concatenated polyproteins based on recombinantly expressed maltodextrin-binding protein (MBP) are shown here to be well-suited for this purpose. Introduction of two specific recognition sites into the primary sequence of the polyprotein allows for the selective cleavage of MBP3 into MBP and MBP2. Moreover, these MBP2 and MBP3 oligomers can be dimerized specifically, such that generation of MPB4 and MBP6 is possible as well. With the set of calibrants presented here, the m/z range of 40-400 kDa is covered. Since all calibrants consist of the same species and differ only in mass, the ionization efficiency is expected to be similar. However, equimolar mixtures of these proteins did not yield equal signal intensities on a detector specifically designed for detecting high-mass molecules.

Ionic liquids as matrices in microfluidic sample deposition for high-mass matrix- assisted laser desorption/ionization mass spectrometry.

Sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) via a microfluidic deposition device using ionic liquid matrices addresses several problems of standard protocols with crystalline matrices, such as the heterogeneity of sample spots due to the co-crystallization of sample and matrix and the limited capability for high-throughput analysis. Since ionic liquid matrices do not solidify during the measurement, the resulting sample spots are homogeneous. The use of these matrices is also beneficial for automated sample preparation, since crystallization of the matrix is avoided and, thus, no clogging of the spotting device can occur. The applicability of ionic liquids to the analysis of biomolecules with high molecular weights, up to ≈ 1 MDa is shown, as well as a good sensitivity (5 fmol) for recombinant human fibronectin, a protein with a molecular weight of 226 kDa. Microfluidic sample deposition of proteins with high molecular weights will, in the future, allow parallel sample preparation for MALDI-MS and for electron microscopy.

High-mass MALDI-MS using ion conversion dynode detectors: influence of the conversion voltage on sensitivity and spectral quality.

With the development of special ion conversion dynode (ICD) detectors for high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the mass-to-charge ratio is no longer a limiting factor. Although these detectors have been successfully used in the past, there is lack of understanding of the basic processes in the detector. We present a systematic study to investigate the performance of such an ICD detector and separate the contributions of the MALDI process from the ones of the ion-to-secondary ion and the secondary ion-to-electron conversions. The performance was evaluated as a function of the voltages applied to the conversion dynodes and the sample amount utilized, and we found that the detector reflects the MALDI process correctly: limitations such as sensitivity or deviations from the expected signal intensity ratios originate from the MALDI process itself and not from the detector.

Mass discrimination in high-mass MALDI-MS.

In high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the accessible m/z range is limited by the detector used. Therefore, special high-mass detectors based on ion conversion dynodes (ICDs) have been developed. Recently, we have found that mass bias may exist when such ICD detectors are used [Weidmann et al., Anal. Chem. 85(6), 3425-3432 (2013)]. In this contribution, the mass-dependent response of an ICD detector was systematically studied, the response factors for proteins with molecular weights from 35.9 to 129.9 kDa were determined, and the reasons for mass bias were identified. Compared with commonly employed microchannel plate detectors, we found that the mass discrimination is less pronounced, although ions with higher masses are weakly favored when using an ICD detector. The relative response was found to depend on the laser power used for MALDI; low-mass ions are discriminated against with higher laser power. The effect of mutual ion suppression in dependence of the proteins used and their molar ratio is shown. Mixtures consisting of protein oligomers that only differ in mass show less mass discrimination than mixtures consisting of different proteins with similar masses. Furthermore, mass discrimination increases for molar ratios far from 1. Finally, we present clear guidelines that help to choose the experimental parameters such that the response measured matches the actual molar fraction as closely as possible.

Studying the fate of non-volatile organic compounds in a commercial plasma air purifier.

Degradation of non-volatile organic compounds-environmental toxins (methyltriclosane and phenanthrene), bovine serum albumin, as well as bioparticles (Legionella pneumophila, Bacillus subtilis, and Bacillus anthracis)-in a commercially available plasma air purifier based on a cold plasma was studied in detail, focusing on its efficiency and on the resulting degradation products. This system is capable of handling air flow velocities of up to 3.0m s(-1) (3200Lmin(-1)), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10Lmin(-1). Mass balance studies consistently indicated a reduction in concentration of the compounds/particles after passage through the plasma air purifier, 31% for phenanthrene, 17% for methyltriclosane, and 80% for bovine serum albumin. L. pneumophila did not survive passage through the plasma air purifier, and cell counts of aerosolized spores of B. subtilis and B. anthracis were reduced by 26- and 15-fold, depending on whether it was run at 10Hz or 50Hz, respectively. However rather than chemical degradation, deposition on the inner surfaces of the plasma air purifier occured. Our interpretation is that putative "degradation" efficiencies were largely due to electrostatic precipitation rather than to decomposition into smaller molecules.

MALDI-MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single-stranded DNA-binding protein.

The Escherichia coli single-stranded DNA binding protein (SSB) selectively binds single-stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB•ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross-linking, combined with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB•ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19,100) can be detected with standard MALDI-MS. With chemical cross-linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79,500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB•ssDNA complex at m/z 94,200 even in the absence of chemical cross-linking. The stability of the SSB•ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB•ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB•ssDNA complex is how ssDNA binds to SSB, rather than the protein-to-DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non-specific aggregation in the MALDI plume.