Determination of phosphorus in small amounts of protein samples by ICP–MS

Abstract

Inductively coupled plasma mass spectrometry (ICP–MS) is used for phosphorus determination in protein samples. A small amount of solid protein sample (down to 1 µg) or digest (1–10 µL) protein solution was denatured in nitric acid and hydrogen peroxide by closed-microvessel microwave digestion. Phosphorus determination was performed with an optimized analytical method using a double-focusing sector field inductively coupled plasma mass spectrometer (ICP–SFMS) and quadrupole-based ICP–MS (ICP–QMS). For quality control of phosphorus determination a certified reference material (CRM), single cell proteins (BCR 273) with a high phosphorus content of 26.8±0.4 mg g^–1, was analyzed. For studies on phosphorus determination in proteins while reducing the sample amount as low as possible the homogeneity of CRM BCR 273 was investigated. Relative standard deviation and measurement accuracy in ICP–QMS was within 2%, 3.5%, 11% and 12% when using CRM BCR 273 sample weights of 40 mg, 5 mg, 1 mg and 0.3 mg, respectively. The lowest possible sample weight for an accurate phosphorus analysis in protein samples by ICP–MS is discussed. The analytical method developed was applied for the analysis of homogeneous protein samples in very low amounts [1–100 µg of solid protein sample, e.g. β-casein or down to 1 µL of protein or digest in solution (e.g., tau protein)]. A further reduction of the diluted protein solution volume was achieved by the application of flow injection in ICP–SFMS, which is discussed with reference to real protein digests after protein separation using 2D gel electrophoresis.

The detection limits for phosphorus in biological samples were determined by ICP–SFMS down to the ng g^–1 level. The present work discusses the figure of merit for the determination of phosphorus in a small amount of protein sample with ICP–SFMS in comparison to ICP–QMS.