Fast and accurate absolute-quantification by ICP-MS

28th September 2015

Application note Proteomics | Quality Assistance

The fruit of a 2 year internal R&D project, Quality Assistance has overcome the challenge of developing and validating a fast and accurate method for absolute-quantification of proteins and antibodies using Isotope Dilution -Triple Quadrupole ICP-MS.

The results were communicated at the European Antibody Congress in Geneva in 2014 and also during a webinar (study sheet and slides available in "Your personal access").

In the light of the interest of the application developed using their technology (ICP-QQQ 8800), Agilent Technologies suggested they publish our results in one of their application notes.
We thank them for their confidence in our expertise and we are very happy to share the application note with them. 

Fast and accurate absolute-quantification of proteins and antibodies using Isotope Dilution-Triple Quadrupole ICP-MS

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is used increasingly in metallomic studies to analyze metals and metal species and their interactions within biological and ecological systems. It is an elemental detection technique that uses a very high temperature argon plasma as the ion source. As a result, a given concentration of an element provides a signal that is broadly consistent, irrespective of the compound structure or sample matrix. Key to achieving accurate and reliable measurements is the ability to reduce and eliminate polyatomic interferences that otherwise would bias the analytical results. Recent development of a triple quadrupole ICP-MS (ICP-QQQ)  dramatically improves the efficiency and reliability of removal of spectral interferences, making it easier to analyze elements with spectral overlaps such as iron, sulfur and selenium. Furthermore, the more effective removal of spectral overlaps allows access to multiple isotopes of many elements, enabling quantification of metalloproteins and peptides through the use of isotope dilution mass spectrometry (IDMS) analysis, which eliminates the requirement for compound specific calibration standards [1]. This absolute quantification technique allows accurate quantification without the need for a reference standard, which is a major benefit of ID-ICP-QQQ for life science research, where many compounds are unknown.

More traditional “absolute” methods of analysis for protein quantification include colorimetry, amino acid (AA) analysis and UV-Vis spectroscopy but each of these techniques has its limitations. Colorimetry, which relies on a color reagent to determine the concentration of a chemical element or chemical, and often requires a Bovine Serum Albumin (BSA) standard, provides poor precision and repeatability. The accuracy and precision achieved with AA analysis is better, although sample preparation involving protein hydrolysis and AA derivatization is time consuming. UV-Vis spectroscopy requires knowledge of the molar extinction coefficient, i.e. prior  availability of the reference protein, and spectral interferences often lead to positive bias. In addition, classical relative methods such as enzymelinked immunosorbent assay (ELISA), LC-MS, LC-UV all require calibration with a standard solution of the protein to be assayed, and such standards are not readily available in most cases. Clearly there is a requirement for a fast, generic method that provides accurate protein quantification without the need for a reference material, in this case a protein standard.

ICP-MS is a trace metals technique, but relatively few proteins contain a significant quantity of a metal as part of their normal composition; examples include hemoglobin (Fe) and superoxide dismutase (Cu/Zn, Mn). However, most proteins (> 95%) do contain sulfur from methionine and cysteine residues [2, 3]. For example, BSA has the formula C2932H4614N780O898S39 and is therefore 1.833% S by weight. Sulfur determination is challenging for conventional quadrupole ICP-MS due to the element’s high ionization potential (10.4 eV) that leads to low sensitivity, and to the occurrence of spectral overlaps from multiple polyatomic ions for all isotopes of S (Table 1), especially in an organic matrix. High resolution ICP-MS (HR-ICP-MS) can be used for the analysis of S, but, apart from the expense of the technique, it suffers a 10-fold reduction in ion transmission efficiency (sensitivity) at the mass resolution required to separate the S isotopes from the spectral interferences.

In this study, we evaluated an Agilent 8800 ICP-QQQ and isotope dilution analysis (ID-ICP-QQQ) of sulfur, for the quantification of NIST BSA 927e standard reference material (SRM), a human immunoglobulin mixture, and a trastuzumab solution. The 8800 ICP-QQQ is ideally suited for IDMS analysis of S as it removes multiple matrix interferences using MS/MS, freeing up the three most abundant S isotopes for accurate measurement.

Authors: Philippe De Raeve, Scientific Director and Juliusz Bianga, Scientist - Quality Assistance s.a.

Application note written for Agilent Technologies.

Release date: September 2015

Download this Application Note