10.2 Biofluid proteomics (plasma, urine, cerebrospinal fluid)

Biofluid proteomics analyzes proteins in bodily fluids using mass spectrometry. It combines protein separation, quantification, and bioinformatics to identify thousands of proteins. This powerful approach offers insights into health and disease states.

Clinical applications of biofluid proteomics are wide-ranging. From cancer biomarkers to cardiovascular risk assessment, this field is revolutionizing diagnostics and treatment monitoring across various medical specialties.

Principles and Techniques in Biofluid Proteomics

Principles of biofluid proteomics

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• Mass spectrometry-based techniques analyze proteins in complex biological samples
  • Bottom-up proteomics digests proteins into peptides before analysis enables identification of large numbers of proteins
  • Top-down proteomics analyzes intact proteins preserves post-translational modifications and protein isoforms
  • Shotgun proteomics uses liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) identifies thousands of proteins in a single run
• Protein separation methods isolate and concentrate proteins prior to analysis
  • Gel-based techniques (2D-PAGE) separate proteins based on isoelectric point and molecular weight visualize protein spots
  • Liquid chromatography (LC) separates proteins or peptides based on physical properties (size, charge, hydrophobicity) improves detection of low-abundance proteins
• Protein quantification approaches measure relative or absolute protein abundance
  • Label-free quantification uses peptide intensity or spectral counting cost-effective for large-scale studies
  • Isotope labeling methods (SILAC, iTRAQ, TMT) incorporate stable isotopes for precise relative quantification across multiple samples
• Bioinformatics tools for data analysis process and interpret complex proteomics datasets
  • Database searching algorithms match experimental spectra to theoretical peptide fragments identify proteins
  • Protein identification software integrates peptide-level data to confidently assign protein identifications and quantifications
• Targeted proteomics techniques focus on specific proteins of interest
  • Selected reaction monitoring (SRM) monitors predetermined peptides and transitions highly sensitive and specific
  • Parallel reaction monitoring (PRM) acquires full MS/MS spectra for targeted peptides improves selectivity and quantification accuracy

Biofluid proteomes for biomarker discovery

• Plasma proteome contains proteins from various tissues and organs
  • High dynamic range of protein concentrations spans over 10 orders of magnitude
  • Rich source of systemic biomarkers reflects overall health status
  • Challenges due to abundant proteins (albumin, immunoglobulins) mask low-abundance proteins
• Urine proteome provides insights into kidney and urinary tract health
  • Non-invasive collection allows frequent sampling and longitudinal studies
  • Reflects kidney and urogenital tract health directly related to renal filtration and secretion
  • Lower protein concentration compared to plasma requires concentration steps before analysis
• Cerebrospinal fluid (CSF) proteome directly reflects central nervous system health
  • Direct reflection of central nervous system health proximity to brain and spinal cord
  • Lower protein concentration and complexity than plasma facilitates detection of brain-specific proteins
  • Potential for neurodegenerative disease biomarkers (Alzheimer's, Parkinson's, multiple sclerosis)

Sample Preparation and Clinical Applications

Challenges in biofluid sample preparation

• Pre-analytical considerations ensure sample quality and reproducibility
  • Sample collection and storage protocols standardize procedures to minimize variability
  • Minimizing protein degradation and modifications use protease inhibitors and low temperatures
• Depletion of high-abundance proteins improves detection of low-abundance proteins
  • Immunoaffinity columns for albumin and IgG removal selectively bind and remove abundant proteins
  • Combinatorial peptide ligand libraries equalize protein concentrations across the dynamic range
• Protein concentration techniques increase sensitivity for low-abundance proteins
  • Ultrafiltration concentrates proteins based on molecular weight cut-offs
  • Precipitation methods (acetone, TCA) concentrate proteins and remove interfering substances
• Protease inhibitors prevent protein degradation during sample handling and storage
• Fractionation strategies reduce sample complexity and increase proteome coverage
  • Strong cation exchange (SCX) separates peptides based on charge
  • High-pH reversed-phase fractionation orthogonal separation to low-pH LC-MS/MS

Clinical applications of biofluid proteomics

• Cancer biomarker discovery improves diagnosis and treatment monitoring
  • Early detection markers in plasma (PSA for prostate cancer, CA-125 for ovarian cancer)
  • Monitoring treatment response and recurrence using protein panels
• Cardiovascular diseases use protein biomarkers for diagnosis and risk assessment
  • Protein panels for myocardial infarction diagnosis (troponin, CK-MB, myoglobin)
  • Risk stratification markers predict future cardiovascular events (C-reactive protein, NT-proBNP)
• Neurodegenerative disorders utilize CSF and plasma biomarkers for diagnosis and monitoring
  • CSF biomarkers for Alzheimer's disease (amyloid-β, tau) reflect brain pathology
  • Plasma markers for Parkinson's disease (α-synuclein, DJ-1) offer less invasive testing
• Kidney diseases employ urine proteomics for early detection and monitoring
  • Urine proteomics for early detection of renal damage identifies subtle changes in kidney function
  • Monitoring transplant rejection uses donor-specific antibodies and injury markers
• Infectious diseases analyze host response proteins for diagnosis and prognosis
  • Host response proteins in sepsis (procalcitonin, C-reactive protein) guide antibiotic therapy
  • Viral infection markers in plasma aid in diagnosis and treatment monitoring (HIV, hepatitis)