Complexity reduction of clinical samples for routine mass spectrometric analysis

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Measuring protein levels in complex biological samples like plasma is challenging due to wide concentration ranges and peptide interferences.
• Existing mass spectrometry (MS) instrumentation advances offer improved selectivity and sensitivity but require optimized sample preparation for robust assays with low limits of detection (LOD).

Purpose of the Study:

• To discuss common protein- and peptide-based fractionation methods for complex biological samples.
• To highlight methods suitable for clinical biomarker validation and routine mass spectrometry analyses.
• To address challenges in precise protein abundance measurement in plasma.

Main Methods:

• Review of protein-based fractionation techniques.
• Review of peptide-based fractionation techniques.
• Discussion of methodologies applicable to routine quantitative studies and clinical samples.

Main Results:

• Various protein and peptide fractionation methods have been developed over two decades to reduce concentration ranges and peptide complexity.
• While many methods are useful for discovery, few are suitable for routine quantitative studies.
• Practical examples illustrate the utility of discussed methods for specific applications.

Conclusions:

• Sample preparation techniques, particularly fractionation, are pivotal for robust, routine mass spectrometric assays in clinical settings.
• Effective fractionation is essential for overcoming challenges in precise protein quantification in complex matrices like plasma.
• The reviewed methods support biomarker validation and routine clinical mass spectrometry.