Macromolecular cystatin C can be a caveat for estimating glomerular filtration rate in biliary obstruction.

heterozygotes, the mean ␤-chain mass was 15 866.832 Da (0.039 Da SD), which is 0.423 Da lower than that determined for the normal ␤-chain. This mass difference is readily detected and even allows the mean abundance of the variant ␤-chain to be estimated as 43.0%, which agrees with the mean abundance determined from the HPLC data associated with each sample (41.0%, 3.2% SD, n ϭ 22). The above SD values imply that mass changes down to between 0.1 and 0.2 Da can be reliably detected. A Ϫ0.2 Da mass change would suggest a heterozygous Ϫ1 Da mutation (Glu3 Lys, Glu3 Gln, Asp3 Asn, or Asn3 Ile) at 20% abundance or heterozygous Lepore-Boston-Washington (normal ␤ Ϫ 2 Da) at 10% abundance. Of course, although ESI-MS of the globin chains can detect Ϯ1 Da mutations, precise identification requires enzymatic digestion. He-moglobins C, D-Punjab, E, and O-Arab can all be positively identified directly from the mass spectra of 30-min digests with trypsin (4). It is still necessary to initially detect zero mass change mutations by electrophoretic or chromato-graphic means, however. Nevertheless , it is useful to know that if HPLC has detected a Ͼ20% abundant variant with a significant polarity change and ESI-MS has not detected a mass change, then Gln7Lys mutations should be considered. We also point out that the minor components, HbA 1c and HbA 2 , are readily quantified by ESI-MS. HbA 1c can be determined from the concentrations of gly-cated ␣-and ␤-chains after calibration with standards (5). Furthermore , we routinely determine HbA 2 (approximately 3%) and find it useful for distinguishing homo-zygous HbE and HbE/␤-thalassemia (HbA 2 and HbE coelute by HPLC). The methods we employ for analyzing Hb have been described in detail (3–5). To reliably achieve the mass measurement precision mentioned above, however, we stress that the data must be acquired over a limited m/z range (we currently scan m/z 930 –1210) with a minimum of 32 data points per m/z unit. Also, each m/z spectrum must be internally calibrated , generally using the multiply protonated ␣-chain ions present from most samples. In this way, inaccuracies due to uncertainties in the atomic weights of the elements are largely eliminated, since the latter are likely to be the same in all proteins in a given sample. We deconvolute m/z 980 –1180 to produce mass spectra using the maximum entropy (Max-Ent)-based software supplied with the mass spectrometer. spectrometry: …