1. Introduction

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was the first time developed by Hillekanmp and Karas [1]. Briefly, the irradiation of a laser with a specific wavelength on a solid sample, covered with a small UV absorbing molecule, leads to the production of gaseous ions, that are analyzed based on their mass-to-charge ratio (m/z) by a mass spectrometer [2]. This technique is applied to several scientific fields from chemistry to biomedicine because can detect a broad variety of compounds such as small molecules until large proteins using a very small amount of sample. MALDI-MS is commonly used in proteomics to identify proteins and their post-translational modifications by detection of tryptic peptides and its comparison to specific databases [3,4]. On the other hand, the soft nature of the MALDI allows also us to analyze entire protein complexes that are bound by noncovalent interactions [5]. Understanding noncovalent interactions between proteins are of great interest and crucial for the biological function such as enzymatic catalysis [6], modulation of signal transduction [7], and immunomodulation [8]. In this regard, the conformation of the protein has a strong effect on the noncovalent interactions and therefore on biological effects [9]. The information regarding the influence of the conformation in the interaction and vice versa is a great interest not only for basic research or structural biology but also in the pharmaceutical fields in which protein-based drugs are continuously developed e.g., vaccines [10]. The interaction of antigen and antibody is a great example of how the conformation of the antigen is crucial for its interaction [11,12]. Therefore, noncovalent interactions are involved in the functionality of a complex protein network [13]. To analyze this complex and sensitive information, sophisticated methods are applied to preserve the conformational and reactivity of the protein, as shown in Figure 1.

2. MALDI Mass Spectrometry Analysis as an Approach to Unravel Interactions in Protein Assembly Guided by Molecular Conformation

Mass spectrometry is a technique able to measure the m/z of several ionized compounds that are analyzed through the use of a mass analyzer and highly sensitive detectors giving several advantages in analytical procedures such as high specificity, sensitivity, speed, and stoichiometry [80,81]. In general, a mass spectrometer consists of three components as schematically shown in Figure 3.

Figure 3. General components of a mass spectrometer. The ions, produced by the ion source, are separated based on their m/z ratio and detected by high sensitive detectors. The ion source produces the ions in the gas phase from the sample. In the mass analyzer, such as triple quadrupole and time-of-flight (TOF), the ions are separated based on the m/z charge ratio and at the end, the ionic currents are detected by conversion to electrical signals in the detector.

The ion source is the main component and the heart of the mass spectrometer in which the analytes are ionized through specific and diverse ionization processes, and therefore detectable by the mass spectrometer. The parameters of the ion source and the sample preparation strongly affect the energy stability of noncovalent interactions, that can be preserved during the ionization process in the gas phase [82]. Therefore, the ion source and its ionization process strongly affect the sensitivity of the noncovalent structures such as the tertiary and quaternary structures of proteins.

Soft ionization processes, such as electrospray ionization (ESI) and MALDI, are employed for structural analysis of the protein elucidating not only noncovalent interactions but also conformational changes of the proteins. For such purpose, several techniques such as hydrogen/deuterium exchange (HDX), native MS, limited proteolysis, cross-linking, and fast photochemical derivatizations are applied to investigate the relationship function and conformation [16,83].

Although Electrospray Ionization (ESI) is the preferred method to study conformations and noncovalent interactions between proteins [84], MALDI-MS remains a simple and fast alternative to study protein-protein complexes. Even though the high energy of the laser, MALDI can preserve noncovalent interactions [5], and yields stoichiometric information of the protein-protein complex [16]. A general advantage of implementing MALDI in analytical procedures is also a user-friendly sample preparation (small sample volumes, no complicated protocols, and high salt tolerance), compatible with high-throughput screening procedures [85] and clinical applications [86]. On the other hand, special and expensive detectors such as ion conversion dynode (ICD), which is the only commercially available high-mass detector, are required for the detection of intact protein-protein complexes with very good sensitivity [87]. Another disadvantage of MALDI-MS to take into account is the challenging in the performance of absolute quantification due to sample composition, sample morphology, inhomogeneous matrix crystallization, laser conditions and, depletions during laser exposure [88]. This review is going to describe MALDI-MS-based procedures for finding functional conformations of a protein involved in a protein-protein complex as described in Figure 4 and all the examples are summarized in Table S1.

Figure 4. Conformation and protein-protein interaction study based on MALDI-mass spectrometry finding the functional conformation.

Limited proteolysis mass spectrometry is a trend method to study conformational effects upon interaction with small molecules [89]. The limited proteolysis is restricted digestion of the protein with a low concentration of proteases. It leads to controlled proteolysis at putative cleavage sites, based on the backbone plasticity and/or local unfolding of the protein region [90].

A combination of limited proteolysis and MALDI-MS was applied for the first time to study a multi-component biomolecular assembly DNA-protein [91] and to perform structural studies using proteolytic footprinting [92]. Similar approaches were applied to protein-protein interactions between cell cycle regulatory proteins, p21 and Cdk2 [93,94], or on capsid viral protein complexes to elucidate mobile features of the viral proteins [95] e.g., autocatalytically cleavable protein α in Flock House virus [96]. The activation-dependent conformational changes of ß-arrestins are revealed using limited proteolysis in combination with both sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analysis. This mass spectrometry-based method can be adapted as a tool to study the nature of ß-arrestins conformational changes downstream of different receptors, as well as to study how each conformational change can be associate with a specific ß-arrestin function e.g., desensitization, internalization, and signaling [97,98]. The structure-function dependent interaction between Hsp90 and its co-chaperone Cdc37 resulting in the facilitation of folding and activation of numerous proteins. Such interaction was studied by limited proteolysis in conjunction with MALDI-TOF-MS analysis of peptide fragments and peptide microsequencing [99]. Another example is given by epitope analysis of antibody-antigen complexes performed also by limited proteolysis coupled with MALDI-MS [100] as shown the Figure 5.

Figure 5. Epitope analysis by limited proteolysis-MALDI-MS approach. Two proteases I and II, which cleaves the antigen at different sites, are used for digestion. The immobilized antibody-epitope complex can be analyzed directly by MALDI-MS. Adapted from [100].

This entry is adapted from the peer-reviewed paper 10.3390/molecules25214979