Mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin α chains in two‐dimensional gel electrophoresis

Abstract

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.

Recent advances in blood‐related proteomics

“…However, several biochemical modifications appear to be superimposed on genetic polymorphisms. Using MS protein structure characterization, the cause of migrational differences of haptoglobin a chains in 2-DE was elucidated [35,36]. It was shown that at least three structurally different protein species can be differentiated and accounted for the most commonly observed spot patterns of haptoglobin a chains.…”

Multifactorial analysis of affinity-mass spectrometry data from serum protein samples: A strategy to distinguish patients with preeclampsia from matching control individuals

“…One sample measurement series (M5) was recorded after fractionated precipitation. Protein mixtures were analyzed with a Reflex III MALDI TOF mass spectrometer (Bruker Daltonik) equipped with the SCOUT source and delayed extraction and operated in linear positive ion mode using an acceleration voltage of 20 kV [25]. Spectra were recorded in a mass range from 4 to 25 kDa and 20 to 250 kDa, respectively, accumulating 900 shots per spectrum.…”

“…Sample preparation of peptide mixtures was performed on an AnchorChip 600/384 target plate [32] using CHCA as matrix. Peptide mixtures were analyzed with a Reflex III MALDI TOF mass spectrometer (Bruker Daltonik) equipped with the SCOUT source and delayed extraction and operated in positive ion mode using an acceleration voltage of 20 kV [25]. Spectra were externally calibrated using a commercially available Peptide Calibration Standard (Bruker Daltonik) as well as internally recalibrated using the following peptide ion signals derived from trypsin autoproteolysis: …”

Presence of free haptoglobin alpha 1S-subunit in acute porcine reproductive and respiratory syndrome virus infection

“…Humans express three different forms of Hp a-subunits, a1S, a1F and a2, that combine with a single b-subunit to generate multimers (Bowman & Kurosky, 1982;Mikkat et al, 2004;Tubbs et al, 2005). Pigs possess one a-subunit, which resembles human Hp-a1S, with one b-subunit (Ponsuksili et al, 2002).…”