Speciation of trace elements in proteins in human and bovine serum by size exclusion chromatography and inductively coupled plasma-mass spectrometry with a magnetic sector mass spectrometer

Abstract

Proteins are separated by size exclusion chromatography while atomic ions from the inorganic elements are detected on-line by inductively coupled plasma-mass spectrometry. A double focusing mass analyzer provides very high sensitivity, low background, and sufficient spectral resolution to separate the atomic ions of interest from most polyatomic ions at the same nominal m/z value. The chromatograms show the distribution of the elements of interest between protein-bound and free fractions and provide the approximate molecular weights of those protein fractions that contain the elements monitored. The distribution of various elements, including V, Mo, Fe, Co, Mn, and lanthanides, in human or bovine serum samples are shown. Alkali metals and Tl are present primarily as free metal ions and are not bound to proteins. Inorganic elements spiked into the serum samples can be followed into various proteins. EDTA does not remove Fe, Pb, Sn, or Th from the proteins but does extract Mn from some proteins. Procedures for determining the effects of breaking disulfide linkages on the metal binding characteristics of proteins are also described.

Measurement of Trace Elements in Proteins Extracted from Liver by Size Exclusion Chromatography-Inductively Coupled Plasma-Mass Spectrometry with a Magnetic Sector Mass Spectrometer

“…1 and 2). Thus, the nebulizer and desolvation system do not broaden the chromatographic peaks greatly, at least for the relatively low-resolution separations obtained by SEC, in agreement with previous work (14,15).…”

“…The column is suitable for separating proteins with molecular weight (MW) from 10,000 to 500,000 Da. This column is larger than those used previously (14,15) and is capable of better separations, but it requires larger samples and has a longer void time. Chromatograms for Ca and Mg showed only small molecules containing these elements, so the shorter, faster column described previously was adequate.…”

“…For the ions studied in the present work, either low or medium resolution was adequate (14,15). The accelerating voltage was nominally 8 kV.…”

“…A different column (GPC 300 at 0.1 ml/min) (14,15) was adequate for the measurement of Ca and Mg. Proteins elute much faster from this smaller column than for the one used for the results shown above. Figure 8 shows chromatograms for Ca and Mg in liver extract.…”

“…Two previous papers from our group describe the use of SEC-ICP-MS with a magnetic sector instrument for analysis of serum and DNA solutions (14,15). Many unusual elements can be measured with very good signal-to-noise ratio without preconcentration procedures, despite the dilution that accompanies the chromatographic separation.…”