Proteomics in Inherited Metabolic Disorders

Abstract

Inherited metabolic disorders (IMD) are rare medical conditions caused by genetic defects that interfere with the body's metabolism. The clinical phenotype is highly variable and can present at any age, although it more often manifests in childhood. The number of treatable IMDs has increased in recent years, making early diagnosis and a better understanding of the natural history of the disease more important than ever. In this review, we discuss the main challenges faced in applying proteomics to the study of IMDs, and the key advances achieved in this field using tandem mass spectrometry (MS/MS). This technology enables the analysis of large numbers of proteins in different body fluids (serum, plasma, urine, saliva, tears) with a single analysis of each sample, and can even be applied to dried samples. MS/MS has thus emerged as the tool of choice for proteome characterization and has provided new insights into many diseases and biological systems. In the last 10 years, sequential window acquisition of all theoretical fragmentation spectra mass spectrometry (SWATH-MS) has emerged as an accurate, high-resolution technique for the identification and quantification of proteins differentially expressed between healthy controls and IMD patients. Proteomics is a particularly promising approach to help obtain more information on rare genetic diseases, including identification of biomarkers to aid early diagnosis and better understanding of the underlying pathophysiology to guide the development of new therapies. Here, we summarize new and emerging proteomic technologies and discuss current uses and limitations of this approach to identify and quantify proteins. Moreover, we describe the use of proteomics to identify the mechanisms regulating complex IMD phenotypes; an area of research essential to better understand these rare disorders and many other human diseases.

Keywords: biomarkers; enzyme replacement therapy; inborn errors of metabolism; lysosomal disorders; proteomics.

Figure 1

“Bottom-up” and “top-down” Proteomic Strategies in clinical samples. In “bottom-up” proteomics approaches, proteins are processed with an exogenous protease (e.g., trypsin), producing internal peptides that have adequate properties for LC MS/MS and database searches, including length, ionization, and fragmentation parameters. These peptides can be analyzed by shotgun proteomics methods to identify the parent protein based on the endogenous cleavage site, and thereby identify the protein itself. By contrast, top-down proteomics permits both strategies: protein cleavage with proteases and direct protein identification without cleavage.

Figure 2

MALDI and ESI Ionization. In MALDI ionization, samples are co-crystallized with an organic matrix on a MALDI plate. A pulsed laser irradiates the co-crystals. This induces rapid heating and desorption of ions into the gas phase. Ions pass through the mass analyzer, such that the smaller peptides reach the detector before the larger peptides (time of flight; TOF). The detector registers the numbers of ions at each individual mass-to-charge (m/z) value, and then the peptide mass fingerprint is generated. In ESI-MS, sample molecules (peptides) are ionized directly in solution. The peptide solution passes through a heated capillary device, and droplets of solution are then sprayed into a vacuum chamber containing a high-strength electric field. The resulting ions pass through a mass analyzer and reach the detector, generating complex spectra with multiply charged ions.

Figure 3

Quantitative methods for proteomics. A quantitative proteomics workflow involves protein extraction/preparation and digestion followed by LC-MS/MS analysis; therefore, there are multiple time points at which peptide quantitation strategies may be introduced. They can be included in cell culture or animal model samples that can be labeled metabolically at the protein level. After cell lysis or protein purification (in clinical samples), labeled proteins can be spiked. During enzymatic digestion, enzymatic labeling can be performed using ¹⁸O. After digestion, peptides can be labeled chemically or isobarically. Finally, label-free techniques can be used: in these cases labeling of samples is not required, and quantitation is performed during or after data analysis.

Figure 4

DDA and DIA mode in proteomic analysis. In DDA mode, extracted proteins are digested and directly analyzed by single-shot DDA. The acquired data is searched against a database of known protein sequences and further processed using diverse software tools. In DIA mode, two distinct forms of proteomic analysis can be performed: SWATH-MS (untargeted DIA) and SRM/MRM (targeted DIA). In SWATH-MS extracted proteins are digested and directly analyzed by single-shot analysis in order to generate the spectral library. Once the library is generated, a multi-window running method is applied to individual samples; wide isolation windows span the entire MS1 m/z range and all precursor ions in the library that are found in each isolation window are fragmented in MS2. All proteins found in the library are quantified (untargeted) and all samples are processed using data analysis software to obtain the quantitative data. In MRM and SRM, the precursor ions to be analyzed are fixed (targeted) by the user, and only these are fragmented in MS2.

Figure 5

Proteomics methods used for the characterization of inherited metabolic disorders. A combination of expression proteomics (quantitative analysis of protein expression across different samples) and functional proteomics (analysis of the properties of molecular networks in a living cell) can be very useful in the search for diagnostic biomarkers and therapeutic targets in IMDs. Moreover, the information acquired using these two approaches can help to better understand the pathophysiology of the disease.