21
Figure 1A. Ion exchange separation of human hemoglobin and mass spectrometry showing predominant alpha and beta chains (unmodifi ed). Part of human haemoglobin mass-spectra showing 1000-1500 m/z range (A0). Six ionisation states for each globin chain are visible. On the insert the ion exchange chromatogram of the initial human haemoglobin preparation.
calculated by the formula: DGIHb
= [A1c(real)-A1c(expected)]/A1c(real),
where A1c(expected) was estimated according to ADA recommendations [2].
Results and Discussion,
Ion exchange chromatography at present provides the highest resolution in analysing haemoglobin heterogeneity. It’s also one of the most suitable methods for the total haemoglobin screening and preparative isolation of single fractions. Electrophoretic methods are less widely used so far in haemoglobin analysis, although they have great potential, especially Isoelectric Focusing (IEF).
Ion exchange chromatography with strong cation exchanger MonoS In the previous studies of haemoglobin
heterogeneity using Mono S cation
exchanger reported by different researchers the buffers used were with a relatively low pH, below pH6, [3]. Under these conditions the different post-synthetically modifi ed haemoglobin isoforms do not obtain suffi cient relative charge differences for optimal separation because the pI differences are not pronounced as they are, when the pH of the running buffer is closer to the isoelectric points (pI) of the proteins of interest. By increasing pH it was possible to increase the resolution. Highest resolution (Figure 1) was achieved in the
pH range 6.6-6.8 which is very close to the pIs of the haemoglobin isoforms of interest (pH7-7.2, approximately). The specifi ed conditions allowed for additional gain in resolution. Here, at least three components of the A0 peak are clearly detected, which normally are non-resolved. (A0 haemoglobin peak is a major haemoglobin that usually considered as unmodifi ed protein). Also, the ‘A1c’ peak appears to be composed in two components and with slower salt gradients it became possible to increase the resolution between them. A very complex pattern of haemoglobin glycation is illustrated by relatively vide ‘chromatographic spectra’ both for glycated and non-glycated haemoglobin fractions and by sophisticated distribution of glycated/non-glycated alpha- and beta chains in single peaks as revealed by MS.
Figure 1B. Boronate affi nity separation of haemoglobin followed by cation-exchange chromatography. Mono S chromatography of haemoglobins present in glycated (PBA+) and non-glycated (PBA-) fractions obtained by boronate affi nity chromatography. Note the marked heterogeneity present in the PBA+ fraction. Cation exchange chromatography at pH 6.8 in acetate buffer, 20mM.
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