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 micro g) or digest (1-10 micro L) 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 micro g of solid protein sample, e.g. beta-casein or down to 1 micro L 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.

Inductively coupled plasma- and glow discharge plasma-sector field mass spectrometry : Part II. Applications

“…Unfortunately, phosphorus and sulfur are known to be elements which are difficult to determine via plasma-based spectrochemistry, since they are characterized by a high first ionization potential, low ionization efficiencies and suffer from overlap with polyatomic ions such as 15 This situation gets even worse in the case of HPLC coupling with organic solvents used as the liquid phase. [311][312][313] For these applications ICP-SFMS is the method of choice because it resolves the spectral interferences from the analyte.…”

“…320 Additionally, the high resolving power and high sensitivity of SFMS-instruments proved of value in particular when dealing with applications where only small amounts of protein are available. 321,322 Phosphorylation of polypeptides was investigated by coupling reversed phase (RP) high performance liquid chromatography to a ICP-SFMS unit as element-specific detector for P. 323 An acetonitrile (ACN)/trifluoracetic acid (TFA) gradient was used for elution, which causes an additional interference originating from the carbon of the mobile phase, which varies proportional with the gradient. Again, all these interferences can be resolved at a mass resolution R ¼ 4000.…”

A high-throughput flow injection inductively coupled plasma mass spectrometry method for quantification of oligonucleotides

“…However, the quantitative determination of phosphorus -as a mono-isotope element at mass 31 u -by a quadrupole-based ICP-MS is hindered by its high first ionization potential (~10.5 eV) and severe spectral interference derived from the sample matrix and laboratory environment. Isobaric interferences such as 15 [20]. This problem can be resolved by the use of a double-focusing sector field ICP-MS [20,21] that has an enhanced mass resolution (m/Δm) greater than 1500; sufficient to separate the interferences from the P signal.…”

“…Isobaric interferences such as 15 [20]. This problem can be resolved by the use of a double-focusing sector field ICP-MS [20,21] that has an enhanced mass resolution (m/Δm) greater than 1500; sufficient to separate the interferences from the P signal. The main drawback of this technique is the high cost of such high resolution ICP-MS instruments.…”

Elemental imaging mass spectrometry of thin sections of tissues and analysis of brain proteins in gels by laser ablation inductively coupled plasma mass spectrometry

“…[16,28] Of special importance in medical research and life sciences is the determination of phosphorus, especially in protein samples. [29] Phosphorylation is thus one of the most important modifications of proteins, and is implicated both in normal physiology and in human disease, carcinogenesis, and aging. [30] The relation of phosphorylation to many neurodegenerative diseases, such as Alzheimer's or Parkinson's disease, has been recognized in a multitude of papers.…”