What is the Omic Hierachy and how do different levels relate to each other?
- Genome - Everything that could happen
- Transcriptome - Organization and control (RNA expression)
- Proteome - Mechanisms (how things work)
- Metabolome/Lipidome - Phenotype (what actually happens)
Omics are dynamic and influence each other like an orchestra where each layer interacts
What is the proteome and why is it important?
The entire set of proteins within a system/cell at a given time under defined conditions. The mechanistic expression of gene expression (RNA)
Key features:
- Highly dynamic, differs cell to cell
- Changes over time
- Reflects the Transcriptome
- Provides accurate image of cellular state
- Proteins play critical roles in structure, function, and regulation
What determines a proteins unique function?
The exact sequence of amino acids determines the protein’s unique 3-D structure, which in turn determines its specific biological function
How many amino acids are used for protein synthesis and what’s special about them?
20 primary amino acids are used for protein synthesis Key points:
- More than 20 different amino acids found in nature
- Only 20 are used for protein synthesis
- The same 20 are used for ALL life forms on Earth
- Classified by structures and properties of side chains
- Leu and Ile are isobaric (same formula, different structure, same mass)
Why is high-resolution mass spec important?
- Uses precise masses (4+ decimal places)
- Can distinguish between compounds
What is scan speed and why does it matter?
How fast the mass analyzer can scan the entire mass spectrum range
Range: 0.5 to 30 scans/sec
Higher number = faster scanning = better for complex samples
Importance:
- Faster scans capture more data points
- Better for LC-MS with changing elution
- More comprehensive coverage
What are the main 6 types of mass analysers?
- Quadrupole (QMF) - Electric field filtering
- Triple Quadrupole (QT) - Targeted analysis
- Time of Flight (TOF) - Speed-based separation
- Quadrupole Time of Flight (QTOF) - Hybrid system
- Ion Trap (IT) - Trapping and sequential release
- Orbitrap - Orbital frequency measuremen
What is the abundance range of proteins in human plasma?
Human plasma contains:
- 289 common proteins typically found
- 4,826 different proteins total
Abundance range spans ~10 orders of magnitude:
- Most abundant: Albumin, immunoglobulins
- Least abundant: Cytokines, growth factors
What are typical protein sizes?
Average protein sizes:
- Eukaryotic proteins: 472 amino acids = 52 kDa
- Bacterial proteins: 320 amino acids = 52 kDa
What are the main steps in shotgun Proteonomics sample prep?
- Extraction - Cell lysis buffer breaks cells, releases proteins
- Quantification - Bradford assay (need to know protein amount for digestion ratio)
- Fractionation - SDS-PAGE gels separate by molecular weight
- Protein digestion - Trypsin converts proteins to peptides (1:20-40 ratio to avoid autolysis)
- Sample cleanup - Salt removal and preconcentration
- LC/MS analysis
What is SDS-PAGE and what can it detect?
SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
Separates proteins by molecular weight
DiGE (Difference Gel Electrophoresis):
- Detects changes in protein modifications
- Band profile and expression changes
- Performed on whole proteins
- Post-translational modifications appear as ‘shift’ in spot position
- Trade-off: Can detect changes but MS needed to identify the modification type
Why is protein quantification important?
- Check extraction yield
- Estimate trypsin needed for digestion (1:20-40 ratio)
- SDS gel wells hold ~20 μg protein
What methods are available to quantify protein?
- Bradford assay (most common)
- Lowry method
- Bicinchoninic acid (BCA) assay
- Fluorescent-based assay
What equation do you use to calculate protein conc fro ma Bradford assay?
Y=mx+c
What are the 4 basic components of a block diagram of a mass spec system?
- Chromatographic separation (LC/GC)
- Ionization (ESI)
- Mass analyzer (MS1 and MS2)
- Detection and data output (GB to TB!)
What is Electrospray Ionization (ESI) and how does it work?
Process:
- Converts liquid samples to gas phase ions - Forms adducts (typically [M+H]⁺ or [M+nH]ⁿ⁺)
- Ions introduced into mass analyzer
- Soft ionization (doesn’t fragment molecules)
Used for:
- Bottom-up proteomics
- Coupled with LC separation
- Peptide analysis
What is peptide fragmentation nomenclature?
Roepstorff-Fohlman / Biemann system:
B ions:
- Extend from N-terminus
- Contain N-terminal portion
Y ions:
- Extend from C-terminus
- Contain C-terminal portion
How does peptide identification work using MS?
- MS1: Measure intact peptide mass
- MS2: Fragment peptide, measure fragment ions (b and y ions)
- In silico comparison:
- Compare experimental spectrum to theoretical database
- Account for differential ionization
- Match fragment patterns - Database matching:
- Identify peptide sequence
- Match back to protein - Calculate protein coverage
What are the 2 main MS modes?
Discovery mode
Targeted mode
What is MALDI and how does it work?
Matrix-Assisted Laser Desorption Ionization
1. Mix protein with matrix solution
2. Spot on sample plate and dry
3. Laser bombards spot (high-energy pulses)
4. Matrix vaporizes, proteins ionize
5. Transfer to qTOF analyzer
Give and advantage and disadvantage of bottom-up Proteonomics?
Advantage: - Complex mixtures analyzed (thousands of proteins simultaneously) Disadvantage: - Long analysis time (90-120 min/sample)
What are PTMs and what is there function?
Chemical modifications after protein synthesis Functions:
- Regulate activity, localization, interactions
- Control enzyme activity
- Protein folding and stability
- Direct proteins to specific locations
- Protein degradation
Give the 8 main types of PTMs and their disease associations
- Phosphorylation - Cancer, metabolic diseases, heart disease
- Glycosylation - Liver disease, autoimmune, inflammation
- Ubiquitination - Autophagy, immunity, aging
- S-nitrosylation - Neurodegenerative diseases (AD, PD, HD, ALS)
- Methylation - Epigenetics, cancer, cardiovascular disease
- Acetylation - Aging, CNS disorders, schizophrenia 7. Lipidation - Infectious disease, neurodegeneration, cancer
- Proteolysis - Protein activation/degradation
