**5.1 Alzheimer's disease**

*Proteoforms - Concept and Applications in Medical Sciences*

**4. Mass spectrometry**

sor ions [73].

isoforms in histone family and combined this separation with RPLC online in the first dimension to better separation and characterization of intact histones [71].

Proteomic experiments, MS based on comprehensive and total characterization of proteoform from a biological system, besides efficient separation, employ a combination of sensitive detection and accuracy of intact proteins. The technology for identification by MS to top-down proteomics has gained impulse. The accuracy of mass spectrometric characterization of polypeptides involves improvement on ionization, fragmentation and detection conditions. Tandem MS can confirm the protein identification based on the daughter ions and characteristics of the obtained peptide map and primary structure, which thereafter provide exact localization of post-translational or other modification sites. Data-independent acquisition (DIA) methods have been alternatively used to analyze proteoforms particularly suited to the study of PTMs [72]. DIA focuses on the identification and quantitation of fragment ions that are generated from multiple peptides contained in the same selection window of several to tens of m/z, that is, the fragmentation spectra of all the peptides are acquired in each cycle time without any preselection of the precur-

The mass spectrometers are compounded basically into a sample inlet, an ion source, a mass analyzer and a detector [74, 75]. Although MS appeared more than a century ago, its application to protein analysis began in the 1990s, because existing ion sources only allowed the ionization and analysis of inorganic molecules and small organic molecules and proteins are not easily transferred to the gas phase and ionized by the size [76, 77]. Advancement of mass spectrometry technology occurred with the new instrumentation ionizer, matrix-assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI) [78–80]. The development of the mass analyzer applied to analyze intact proteins contributed to the mass spectrometry identification of the proteoforms. Mass analyzers with a high level of resolving power and sensitivity as time-of-flight (TOF), Orbitrap, Fourier Transform Ion Cyclotron Resonance (FT-ICR), or the combination of multiple mass analyzers in series, created a powerful tool for top-down MS characterization of proteoforms [78, 81, 82]. Most top-down proteomics (TDP) studies have used some form of tandem-MS fragmentation techniques, for intact proteins sequencing with greatly resolving power and high mass accuracy as: collisionally activated dissociation (CAD), collision-induced dissociation (CID), electron transfer dissociation (ETD), electron-capture dissociation (ECD), higher-energy collisional dissociation (HCD), infrared multiphoton dissociation (IRMPD) and ultraviolet photodissociation (UVPD). These examples of fragmentation strategies can provide additional information on the amino acid sequence and PTMs for identification of proteoforms [74, 75, 79, 83]. The mass spectrometer sample introduction can be through the traditional RPLC-MS, by CZE-MS or embedded in a matrix on a target plate [74, 84]. Mass spectrometers that use different types of analyzers for the first and second stages of mass analysis (hybrid MS instruments) are employed to maximize proteoform characterization top-down MS-based. Still, software tools for the identification and quantification of proteoforms need to be continuously developed to keep up with a demand to quickly and automatically analyze the data generated. Many a comprehensive proteoform software tools for proteoform identification and construction of proteoform families are freely available: MASH Suite, MetaMorpheus, MSPathFinder, Proteoform Suite, TDPortal, TopMG and TopPIC

**16**

In the diagnosis of Alzheimer's disease (AD), quantification of total Tau protein (T-tau), threonine-phosphorylated Tau181 form (P-Tau181), and the 42 amino acid peptide, alpha-amyloid isoform (Aβ) are well established as markers present in cerebrospinal fluid (CSF). However, there is a constant need for new diagnostic markers to identify the disease at a very early stage [87]. A review about the role of proteoforms in the pathophysiology process of Alzheimer's disease was described in [88]. The mass spectrometry performance of three canonical proteins, clusterin, secretogranin-2, or chromogranin A, was presented. Variations on the levels of Apo A-1, a protein with antioxidant and anti-inflammatory properties, in the serum or in CSF, are also indicated as a potential marker for AD diagnosis and progression. Apo A-1 exhibits [86] and inhibits the aggregation and neurotoxicity of an amyloid-β peptide in AD [89]. The possible association between apolipoproteins increased Apo A-1 levels that were correlated with decreasing risk of dementia [87], raising the possibility of a novel role of Apo A-1 in protection against neurological disorders [87, 89].
