Direct analysis of α- and β-chains of hemoglobins from mammalian blood samples by nanoESI mass spectrometry during in-capillary proteolytic digestion
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α- and β-chains of hemoglobins derived from several species were analyzed directly from diluted blood samples by simultaneous in-capillary proteolytic digestion and nanoESI MS and MS/MS analysis. Starting from fresh or frozen and thawed blood samples, sequence coverages of >80% were usually obtained. Only 2 h after resuspension of a dried blood spot, human origin could be demonstrated from data obtained by in-capillary tryptic digestion, nanoESI mass spectrometric analysis, and data base search. A fast and facile differentiation of closely related species by hemoglobin-derived proteolytic “marker peptides” was demonstrated for Asian (Elephas maximus) and African elephants (Loxodonta africana). Finally, amino acid sequences deduced from collision-induced dissociation experiments during in-capillary proteolytic digestion of the corresponding blood samples allowed de novo sequencing of previously unknown sequences of hemoglobin chains of the Patagonian cavy (Dolichotum patagona) and the Persian gazelle (Gazella subgutturosa subgutturosa). 100% of the α-chain sequences and more than 85% of the β-chain sequences were covered for both the species. Additionally, sequence data derived from tandem MS experiments obtained with the Q-Tof analyzer were confirmed by high resolution Fourier-transform ion cyclotron resonance mass spectrometric experiments. Accurate protein mass determination of the intact hemoglobin chains directly from the corresponding blood samples by use of a Fourier-transform ion cyclotron resonance mass spectrometer corroborated the deduced sequences of the respective α-chains. The present study demonstrates that in-capillary digestion allows fast characterization and/or sequencing of hemoglobin chains directly from blood samples.
KeywordsHemoglobin In-capillary digestion NanoESI MS FT ICR MS De novo sequencing
- Kleinschmidt T, Sgouros JG (1987) Hemoglobin sequences 108. Biol Chem H-S 368:579–616Google Scholar
- Nakanishi T, Kishikawa M, Shimizu A, Hayashi A, Inoue F (1995) Assignment of the ions in the electrospray ionization mass spectra of the tryptic digest of the non-derivatized globin, covering the whole sequence of alpha- and beta-chains: a rapid diagnosis for haemoglobinopathy. J Mass Spectrom 30:1663–1670CrossRefGoogle Scholar
- Patrinos GP, Giardine B, Riemer C, Miller W, Chui DHK, Anagnou NP, Wajcman H, Hardison RC (2004) Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. Nucleic Acids Res 32(Database issue):D537–D541PubMedCrossRefGoogle Scholar
- Takátsy A, Böddi K, Nagy L, Nagy G, Szabó S, Markó L, Wittmann I, Ohmacht R, Ringer T, Bonn GK, Gjerde D, Szabó Z (2009) Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography. Anal Biochem 393:8–22PubMedCrossRefGoogle Scholar