Agilent ICP-MS Journal (May 2006 – Issue 27)

Importance of the Topic

Metalloproteins play vital roles in human physiology, accounting for roughly one third of all proteins that incorporate metal ions for catalysis, transport and storage. Trace detection of metals bound to serum proteins provides insights into metal homeostasis and potential disease biomarkers. Coupling size exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS) enables element-specific profiling across molecular weight fractions in complex biological fluids with minimal sample preparation.

Objectives and Overview of the Study

The study aimed to establish a robust SEC-ICP-MS workflow for pooled human serum to:

• Separate serum proteins by size into high-molecular-weight, mid-range and low-mass peptide fractions
• Detect and quantify nine biologically relevant isotopes at sub-ppb levels
• Demonstrate the potential of the technique for broad‐scale elemental profiling and biomarker discovery

Methodology

Pooled undiluted Type O human serum was injected (10 µL) onto a TSKGel SEC column. A high-ionic-strength 0.1 M sodium acetate mobile phase at pH 7.0 flowed at 0.7 mL/min, limiting charge-based retention. Protein elution was monitored by UV absorbance at 240 nm, and the eluate was introduced via PEEK tubing into an ICP-MS for multi-element detection. Nine isotopes including P, S, Se, Si, and two Fe isotopes were recorded to generate molecular weight–resolved elemental profiles.

Instrumentation Used

• Tosoh Bioscience TSKGel SEC column (5 µm, 25 nm pore)
• Agilent 1100 Series HPLC with Peltier-controlled cooling
• Agilent 7500ce ICP-MS with OpalMist concentric PFA nebulizer
• Agilent ChemStation ICP-MS software for system control and data acquisition

Main Results and Discussion

Three distinct protein size fractions were resolved: large globular proteins (>83 kDa), high-abundance transferrin and human serum albumin (20–83 kDa), and low-mass peptides (<20 kDa), each binding multiple elements. All nine isotopes appeared in at least one fraction. Within the peptide region, three sub-fractions were partially resolved; however, the ~14.7 kDa region showed overlapping peaks for 60Ni and 66Zn, indicating the need for higher chromatographic resolution.

Benefits and Practical Applications of the Method

• Rapid screening of undiluted serum with minimal sample handling
• Element-specific molecular size distribution offers complementary data to proteomic approaches
• Potential to identify and quantify metalloprotein biomarkers for disease diagnosis and monitoring

Future Trends and Potential Applications

Advances in high-resolution SEC and capillary chromatography interfaces will improve separation of closely eluting peptide species. Integration with tandem mass spectrometry for peptide sequencing can link elemental profiles to protein identities. Extending the approach to other biological fluids and tissues may uncover novel metalloprotein biomarkers across a range of diseases.

Conclusion

SEC-ICP-MS provides a powerful, sensitive platform for size-resolved elemental profiling of human serum. The method reliably separates major protein and peptide fractions and detects multiple trace elements, highlighting its value for metallomics research and biomarker discovery. Further enhancements in chromatographic resolution and coupling to proteomic identification techniques promise a more comprehensive understanding of the metalloproteome.

References

• Haraguchi H Journal of Analytical Atomic Spectrometry 2003 19 5–14
• Wind M Wolf DL Journal of Analytical Atomic Spectrometry 2004 19 20–25
• Jakubowski N Lobinski R Moens L Journal of Analytical Atomic Spectrometry 2004 19 1–4
• Lobinski R Szpunar J Analytica Chimica Acta 1999 400 321
• Dudev T Lim C Chemical Reviews 2003 103 773–787
• Coni E Bocca B Galoppi B Alimonti A Caroli S Microchemical Journal 2000 67 187
• Nischwitz V Michalke B Kettrup A Journal of Analytical Atomic Spectrometry 2003 18 444–451
• Shiobara Y Yoshida T Suzuki KT Toxicology and Applied Pharmacology 1998 152 309
• Gallicchio L Flaws JA Sexton M Ioffe OB Toxicology Letters 2004 152 245
• Boulyga SF Loreti V Bettmer J Heumann KG Analytical and Bioanalytical Chemistry 2004 380 198
• Richarz AN Bratter P Analytical and Bioanalytical Chemistry 2002 372 412
• Profrock D Leonhard P Ruck W Prange A Analytical and Bioanalytical Chemistry 2005 381 194