Brain Proteomics

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use of MS systems has caused changes in the intended use of these methods.

For example, gel systems, which were previously widely used in quantitative

protein detection, are now preferred to reduce protein complexity before MS

analysis, while antibody-based methods such as ELISA and western blot are

used to confirm the findings obtained after MS analysis.

There are two main approaches to proteomics: top-down and bottom-up,

which differ depending on the stage of preparation of samples before MS anal-

ysis (Figure 10.1). Among these approaches, shotgun (bottom-up) proteomics

has a wide application for neurodegenerative diseases. Therefore, the focus of

this chapter will be on liquid chromatography-mass spectrometry-based (LC-

MS) methods from the perspective of shotgun proteomics. In the shotgun pro-

teomics workflow, peptides are first obtained from extracted proteins through

denaturation, alkylation, and enzyme digestion. After this step, the peptide

mixture is usually separated by high-performance liquid chromatography and

ionized via an ion source integrated into the MS. Although there are several

techniques for ionization of peptides, the best known are ESI (Electrospray

ionization) and MALDI (Matrix-assisted laser desorption ionization), and ESI

is used combined with liquid chromatography for peptide separation in shot-

gun proteomics. With the ESI technique, ionized peptides are sprayed into a

high-resolution tandem mass spectrometer to measure the mass/charge (m/z)

ratios followed by their fragmentation in the collision cell for the acquisition

of mass spectra.

Finally, raw data files of several GB in size are processed by loading

them into software programs that use various algorithms for peptide-spectrum

matching. Global analysis of proteins by the high-resolution mass spectrom-

etry provides us with information not only about the expression differences

of proteins, but also the amino acid sequence of that protein and what mod-

ifications there are on it [15–17]. There are variety of methods used in this

approach, each with their pros and cons (Figure 10.2). Therefore, each step

in these methods requires meticulous work.

10.5

Processing and Visualizing Proteomics Data

Nowadays, the shotgun proteomics approach offers the opportunity to si-

multaneously examine over ten thousand proteins in brain tissue and over

1000 proteins in cerebrospinal fluid [18, 19]. In this process, successful

sample preparation and LC-MS/MS analysis is followed by processing of

large amounts of proteomic data. However, processing and interpretation

of data is a critical step of the proteomics workflow and is as labor in-

tensive as the other steps. At this step, programs using special software

are needed to process several GB of data obtained by LC-MS/MS analy-

sis. Progenesis QI (Waters), Protein Lynx Global Server (Waters), MaxQuant