22 February / March 2019
Figure 1C. Mass-spectrometry analysis of globin chains of A1c and A0 peaks from ‘Non-Glycated, PBA-‘ and ‘Glycated, PBA+’ fractions, obtained by boronate affi nity chromatography. The left and right panels represent the A0 and A1c peaks obtained by cation-exchange chromatography analysis of PBA- and PBA+ fractions. Only a part of mass-spectrogram is shown: alpha and beta chains with ionisation states: +14a, +15b and +15a, +16b. The modifi ed (Mw-shift of 160 Da, approx.) and unmodifi ed globin chains are marked by the chain symbol with right superscripts ‘X’ or ‘0’, respectively.
Capillary Isoelectric
Focusing with whole column imagine detection (WCID).
Capillary electrophoresis with WCID provides essential advantage in analysis of complex biological mixtures due to its opportunity to observe the whole separation path. Since most traditional CE devices are equipped with just a narrow window at the very end of the capillary, the stage of so-called mobilisation is required to scan a steady-state distribution achieved in IEF process. The latter essentially reduces the resolution and can even potentially result in artifi cial peaks appearing. Figure 2 shows an electropherogram of human glycated haemoglobin. Relatively high resolution, comparable to ion exchange separation, is achieved due to narrow range carrier ampholytes (CAs) preparations. Further improvement in resolution can be connected with using of modifi ed pH gradients- the gradients containing higher number of CA species per pH unit.
Preparative Gel Electrophoresis.
Agilent OFFGel fractionator employs Immobilised pH gradients (IPG) for isoelectric focusing. Due to its original design the sample fractions can be easily collected at the end of experiment. The absence of CA chemicals makes it possible to analyse collected protein fractions without further purifi cation. The OFFGel fractionation combined to MS, perhaps, is the easiest way for the total haemoglobin/ glycated hemoglobin analysis with the possibility of detecting minor components, Figure 3.
Clinical applications: A1c tests comparison and standardisation
The heterogeneity of glycated haemoglobin was fi rst observed in the nineteen fi fties, although it was only with MS that it became possible to give a reasonable qualitative defi nition to A1c [4,5]. A1c was deemed to
be broadly a ‘major’ component of glycated haemoglobin population with a strong belief that represents N-terminal b-chain fructosyl valine. Recently, IFCC has come with a reference method that gives precise defi nition to A1c [6]. The quantitation is based on a MS reference method, and despite some uncertainties associated with the digestion procedure, allows a degree of confi dence in the fi nal data produced. Still, even the IFCC defi nition describes essentially heterogeneous population of glycated haemoglobin (a limited number of glycated haemoglobin ‘isomers’), although not all researches do realize this. The above heterogeneity is connected with the fact of the small glycated hexapeptide originates from a number of different dimer combinatory (a-chain plus b-chain), each of which can be differently glycated on different possible glycation sites. The same can be said about modern Immunoassays: they, in theory, should determine exactly the same population of glycated haemoglobin, provided one can neglect cross reactivity.
The MS detection based on haemoglobin
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
