Mass spectrometric epitope mapping has become a versatile method to precisely determine a soluble antigen's partial structure that directly interacts with an antibody in solution. Typical lengths of investigated antigens have increased up to several 100 amino acids while experimentally determined epitope peptides have decreased in length to on average 10-15 amino acids. Since the early 1990s more and more sophisticated methods have been developed and have forwarded a bouquet of suitable approaches for epitope mapping with immobilized, temporarily immobilized, and free-floating antibodies. While up to now monoclonal antibodies have been mostly used in epitope mapping experiments, the applicability of polyclonal antibodies has been proven. The antibody's resistance towards enzymatic proteolysis has been of key importance for the two mostly applied methods: epitope excision and epitope extraction. Sample consumption has dropped to low pmol amounts on both, the antigen and the antibody. While adequate in-solution sample handling has been most important for successful epitope mapping, mass spectrometric analysis has been found the most suitable read-out method from early on. The rapidity by which mass spectrometric epitope mapping nowadays is executed outperforms all alternative methods. Thus, it can be asserted that mass spectrometric epitope mapping has reached a state of maturity, which allows it to be used in any mass spectrometry laboratory. After 25 years of constant and steady improvements, its application to clinical samples, for example, for patient characterization and stratification, is anticipated in the near future. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:229-241, 2018.

Haptoglobin belongs to the major constituents of plasma and acts as hemoglobin-binding and acute-phase protein. Due to the occurrence of three major allelic variants and further structural modifications, the alpha chains of haptoglobin form varying spot patterns in two-dimensional gel electrophoresis (2-DE) gels, which is generally observed in differential proteome analyses using plasma or related body fluids of humans. In the present study plasma samples from 10 donors of initially unknown haptoglobin phenotype were separated by 2-DE and tryptic digests of excised haptoglobin alpha chain spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-quadrupole ion trap TOF-MS. Haptoglobin alpha1S, alpha1F, as well as alpha2 chains were found to occur each with at least three structurally differing protein species: (i) the unmodified form, which corresponds to the sequence database entries; (ii) derivatives, in which asparagine at position five is deamidated to aspartic acid; and (iii) derivatives with an additional C-terminal arginine residue. These structural variants account for the most commonly observed spot patterns of haptoglobin alpha chains in Coomassie-stained gels. Additionally, a minor derivative of the haptoglobin alpha2 chain carrying both modifications, deamidation at position five and the C-terminal arginine residue, was identified. Theoretical pI values of the characterized structural variants are, consistent with their observed migration in the 2-DE gels.

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Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.

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