Proteomics - Lecture Notes

Flashcards cover key proteomics concepts from sample prep to advanced MS instrumentation, data analysis, quantification, PTMs, interactions, spatial proteomics, single-cell proteomics, and proteogenomics.

What are the four main steps in proteomics sample preparation?

1) Tissue/cell collection and homogenization; 2) Cell disruption; 3) Protein extraction and removal of interfering substances; 4) Protein solubilisation and stabilisation.

What is Dissection as a method for tissue collection?

Physically isolating the organ or tissue; remove fat/connective tissue and surrounding material to reduce contamination; standard for animal/plant tissue studies.

Name three cell disruption techniques used in proteomics.

Osmotic/detergent lysis, sonication, grinding with liquid nitrogen (also rotor-stator high-pressure homogenization and bead beating as options).

What is the purpose of using detergents in cell disruption/extraction?

Detergents associate with exposed hydrophobic regions to prevent protein aggregation and help solubilize proteins.

What buffer components are typically used in protein extraction?

A buffer near physiological pH (e.g., Tris-HCl ~7.5), chaotropes (e.g., urea, guanidine), detergents (e.g., CHAPS), and often DNase; salts may be present and can interfere with MS if not removed.

Why are chaotropes used in protein extraction, and what is a potential drawback?

They disrupt hydrogen bonding and the hydrophobic effect to denature proteins, aiding solubilization; drawback: they can denature proteins and affect downstream analyses.

Name two common protein precipitation methods used to enrich the protein fraction.

Cold acetone precipitation and Trichloroacetic acid (TCA) precipitation.

What is Ultrafiltration used for in protein sample preparation?

Concentrates proteins and removes small molecules from the extract.

What is the purpose of protein subfractionation in proteomics?

To reduce sample complexity by enriching specific subproteomes or depleting abundant proteins, enabling deeper proteome coverage.

Give two examples of major serum protein depletion kits mentioned in the notes.

SERVA BluePrep Major Serum Protein Removal Kit; Thermo TOP14 Top14 Abundant Protein Depletion Resin.

What is MudPIT and why is it used?

MudPIT (Multidimensional Protein Identification Technology) combines multidimensional chromatography (e.g., SCX + RP-LC) before MS to handle complex mixtures.

What does 2D-LC stand for and how does it work?

Two-Dimensional Liquid Chromatography; combines two chromatography modes (e.g., SCX or HILIC in dimension 1 with RP-LC in dimension 2) to improve separation.

What is spectral counting in label-free proteomics?

Counting MS/MS spectra matched to peptides of a protein as a proxy for relative abundance.

What is emPAI and how is it used?

Exponentially Modified Protein Abundance Index; normalizes observed peptide counts by the number of observable peptides to estimate abundance.

What is Area Under the Curve (AUC) in label-free quantification?

Integration of peptide signal across the LC elution profile to quantify abundance; more accurate than spectral counting but data-intensive.

Name common label-based quantitative proteomics methods.

ICAT, cICAT, iTRAQ, TMT, SILAC, Super-SILAC, and related isotope-labeling strategies.

What is AQUA in quantitative proteomics?

Absolute quantification using synthetic labeled peptide standards spiked into samples to obtain absolute amounts.

What does SRM/MRM stand for and what is it used for?

Selected Reaction Monitoring / Multiple Reaction Monitoring; targeted quantification of peptides with high specificity using a triple quadrupole mass spectrometer.

What is PRM and how does it differ from SRM/MRM?

Parallel Reaction Monitoring; targeted proteomics on high-resolution analyzers (e.g., Orbitrap/TOF) that measures multiple fragment ions for each precursor, offering higher resolution and post-acquisition flexibility.

What is Data-Independent Acquisition (DIA) in MS?

An acquisition strategy that fragments all ions within predefined m/z windows, providing comprehensive, unbiased data for identification and quantification.

What is Data-Dependent Acquisition (DDA)?

An acquisition strategy where the instrument selects the most intense ions for MS/MS for fragmentation and analysis.

Name three database search tools used for peptide/protein identification.

Mascot, Andromeda (with MaxQuant), Sequest.

What is Mascot and what scoring approach does it use?

A database search engine that uses MOWSE probability-based scoring to estimate the chance of random matches.

What is Andromeda and how is it commonly used?

A database search engine integrated with MaxQuant, offering refined scoring and PTM tolerance; improves identification and integrates with LFQ.

What is Sequest and its key scoring concept?

An early database search algorithm that uses XCorr scoring to compare experimental MS/MS spectra with theoretical spectra.

What is Ionbot?

ML-based predictive model for peptide fragmentation; supports open modification/mutation searching; high sensitivity but computationally intensive.

What is de novo sequencing in proteomics?

Determining peptide sequences directly from MS/MS spectra without relying on a protein database.

What is spectral library searching?

Matching experimental MS/MS spectra to a library of previously identified spectra for fast, confident identifications when the spectrum exists in the library.

How is the decoy method used in FDR estimation?

Scrambles the target database to create decoys; the rate of decoy matches estimates false discovery rate (FDR) in identifications.

What is MudPIT workflow in terms of chromatography steps?

Protein digestion -> multidimensional chromatography (SCX then RP-LC) -> MS/MS analysis.

What does SCX and RP-LC stand for and what properties do they separate peptides by?

SCX separates by charge (strong cation exchange); RP-LC separates by hydrophobicity (reversed-phase).

What is the concept behind 2D-LC in proteomics?

Two sequential LC steps with orthogonal separation properties to reduce sample complexity and improve peptide separation.

What is the difference between iTRAQ and TMT labeling?

Both are isobaric, multiplexed peptide labeling; iTRAQ uses 4–8-plex and TMT ranges up to 16-plex; both enable peptide-level quantification in MS/MS.

What is SILAC and when is it used?

Metabolic labeling with heavy amino acids in cell culture; proteins incorporate labels and samples are mixed before MS; accurate relative quantification; not suitable for tissues.

What is a carrier proteome in SCoPE-MS?

A large set of pooled cells added to single-cell samples to boost signal while preserving single-cell quantification via barcoding (e.g., TMT).

What is proteogenomics?

Integration of proteomics with genomics and transcriptomics to improve gene annotation and validation of predicted proteins, splice variants, and PTMs.

What is N-terminomics/COFRADIC used for?

Identifies neo-N-termini and translation initiation events, revealing proteoforms and N-terminal modifications.

What is a localisome in spatial proteomics?

A concept describing the spatial proteome; mapping protein localization within subcellular compartments or tissues using imaging or MS-based methods.

What are the major approaches to study protein-protein interactions (PPIs)?

Y2H (Yeast Two-Hybrid), AP-MS/Co-fractionation, and proximity labeling methods (BioID/TurboID/APEX), plus XL-MS.

What is the aim of single-cell proteomics (SCP)?

To measure proteomes at single-cell resolution to capture cellular heterogeneity not seen in bulk analyses.

What is a volcano plot and how do you interpret it?

A plot with x-axis as log2 fold change and y-axis as -log10(p-value); points far to the right/left with high y-values indicate strong, significant changes.

What is CyTOF and what does it measure?

Mass cytometry using metal-tagged antibodies to quantify many proteins per cell (often >40) at single-cell resolution for immune mapping.

What are the main advantages of Orbitrap and FTMS instruments?

Orbitrap offers high resolution and accuracy at lower cost than FTICR; FTMS (including FTICR) provides the highest resolution and mass accuracy but at high cost.

Why is TOF MS often coupled with a reflectron?

To correct for initial energy spread and improve mass resolution in Time-of-Flight analyzers.

What are the advantages and limitations of MALDI vs ESI ionization methods?

MALDI: laser desorption/ionization with a matrix; good for simpler mixtures and high mass peptides; ESI: spray ionization at atmospheric pressure, generates multiple charged ions and integrates well with LC; better for large, complex samples.

What is the typical optimal spray condition for ESI in LC-MS?

Approximately 50% organic solvent (MeOH or CH3CN) with 0.1% formic or acetic acid.

What is the role of imaging mass spectrometry and CyTOF in spatial biology?

Imaging MS maps tissue/organ distribution of peptides/proteins; CyTOF enables multiplexed protein localization in single cells via metal-tagged antibodies.

What is the purpose of proteogenomics resources like OpenProt or IGV in proteomics?

To integrate proteomic data with genomic annotations, validate predicted ORFs, and explore novel protein products and transcript variants.