Proteomics

• Proteomics is the large-scale study of proteins: expression, structure, functions.

• There is one genome but different proteomes from the same genome. The proteome is the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or system. This will vary with time and distinct requirements, or stresses that a cell or organism undergoes

• Contex: in order to understand biological systems at the molecular level, we must analyze proteomes quantitatively, in time and space and under different (patho-) physiological conditions; molecular constituents of biological systems do not operate in isolation, thousands of interactions e.g. mitosis 

• in gel digesion: typically performed with samples containing detergents, high amounts of salts

  1. Sample reduction and alkylation (DTT) 2. SDS PAGE (desalting) 3. Cut out protein bands 4. Wash Gel and let it dehydrate 5. Reswell in protease solution and digest 6. Extract peptides 

  + Simple to perform (scalabe to high troughput)

  + tolerant to samples with "weird" buffer ingredients (e.g. detergents, lipids, DNA)

  + Isolation of proteins of interest possible (single bands)

  - sample amount limited to gel capacity

  - limited visual sensitivity

  - lower peptide recovery

• Ion pair reverse chromatography is a form of liquid chromatography: has the highest resolution

• hydrophobic and electrostatic interactions 

• The stationary phase is highly hydrophobic, the mobile phase starts with being hydrophilic and increases in hydrophobicity with the time. The elutes are hydrophilic in the beginning and hydrophobic in the end 

• Interactions: direct hydrophobic interactions between nonpolar peptide side chains and the nonpolar stationary phase & electrostatic interactions between polar side chains of amino acids in a peptide and an amphiphilic ion pairing agent (e.g. triufluoro-acetic acid) that mediates interactions with the nonpolar stationary phase 

• MALDI = Matrix Assisted Laser Desorption Ionization

• The matrix traps laser energy for desorption (absorption maximum ideally at laser wavelength), protects the analytes from thermal decomposition (hot and cold matrices) and ionises analyte molecules 

• Soft ionization technique because matrix absorbs the energy of the laser and hleps to transfer it to the analyte causing it to vaporize and ionize gently without too much fragmentation of the peptides.

• Soft ionization from the condensed phase (crystals)

MALDI and ESI are both soft ionization methods because they generate gas-phase ions without causing significant fragmentation or degradation of the analyte molecules.

In MALDI, the analyte is mixed with a matrix material that absorbs laser energy and facilitates desorption and ionization of the analyte: Most of the deposited energy by the laser is taken up by the matrix, matrix gets heated, desorption of matrix and material into the vaccum, rapid cooling of evaporized material prevents thermal decomposition. The resulting ions are mainly intact and carry a single charge.

• TOF: Analytes (ions) are accelerated with the same voltage (all start at the same kinetic energy) and then travel through a field-free tube (no force applied). The time it takes for an ion to travel from the ion source to the detector (their velocity vary depending on the m/z) is used to determine its m/z value. t_d = C * sqrt(m/z)
  • Resolution increased by reflection mode TOF and by fast detector digitizer
  • TOFs can separate ions at a rate of >10.000 spectra/second

characteristic: scan speed 100µsec, m/z range > 500000, resolution >20.000, high sensitivity

Quadrupole: Consists of two positive and two negative poles arranged in corners of a square. RF (radiofrequency) amplitude and DC (direct current) voltages are applied to create an oscillating electric field that allows only ions with a specific m/z ratio to pass through the analyzer (Lower amplitude, ions with lower m/z pass). The ions that pass through the quadrupole analyzer are detected by a detector, which records the intensity of the ion signal. The ion intensity data is then used to generate a mass spectrum.

• Resolution depends on the number of oscillations along the path -> resolution increases with longer rods or decreasing scan speed 
• m/z range limitations (up to 4-8000 m/z)

characteristic: dynamic range, also high accuracy, quantification

For the analysis of an intact antibody, I would choose the TOF mass analyzer because it has high resolution, which is important for distinguishing between different antibody isoforms with slight mass differences.

Quadrupole, TOF, Ion trap, Oritrap

• Mass accuracy: 100-1000, 1-10, 100-1000, 1-2 ppm
• resolution: 200-2.000, >20.000, 200-20.000, >100.000
• m/z range: 4-8000, >500000, 4000, 4-8000
• scan speed: 1-10msec, 100µsec, 10-100msec, 20-200 msec
• dynamic range: 1:10000,1:5000, 1:1000, 1:5000
• senstitivity: ++, +++, +++, ++
• quantification: +++, ++, +, ++

• 2 consecutive MS: MS1 one shows m/z of tryptic peptide. The mass spectrometer selects the (20) most abundant ions from MS1 for MS2 -> peptide isolation and fragmentation -> measurement of fragments m/z

• Sequence determination through specific amino acid mass, step by step. Or database searching, whole peptide or sequence tag

• dynamic exclusion prevents the same peptides to be analysed again and again

• Through target-decoy approach: you have 2 databases, one composed of targets and other of decoys. Then search the tandem mass spectra against both databases. The PSMs to decoy sequnces must be wrong matches (false positives). The FDR is determined by FDR = FP / FP + TP = decoys / targets. Then, define a cut-off score (=how many FP you will accept): FDR(x) = decoys with score > x / targets with score > x

• No isotopic label required, no artifitial chemical modification of peptides or proteins: LFQ intensity describes the integrated MS signal (area under the peak) across the chromatographic peak. Therefore, the same peptide is detected in consecutive MS1 spectra. The chromatographic peak is reconstructed based on MS1 signal -> extracted ion chromatogram (XIC). The area under the XIC is a measure for peptide quantity. Relative quantification of the same peptide vs a reference sample or between x conditions. 

• Pros:
  • Cost and time effective with respect to sample preparation (no need to label/ pre-process the samples)
  • Applicable to all identified/ quantified proteins – proteome-wide
  • Applicable to all organisms/ tissues/ cells – independent of biology
  • Applicable to very large numbers of sample – large scale
  • No increase in sample complexity – high sensitivity
• Cons:
  • Const and time ineffective in terms of MS measurement time (individual sample prep and measurement, no multiplexing)
  • Error- prone: Reproducible sample preparation, chromatography and mass spectrometric performance is critical for this approach! Accumulation of experimental variation along the workflow
  • Quantitative precision and accuracy of label free analysis are often lower than those based on stable isotope labeling, particularly when workflow comprises many steps
  • Missing values across different sample increases with increasing number of samples
  • (Large-scale label-free projects are computationally intensive and large bioinformatics resources may be needed

• DDA (shotgun) data is analysed without prior knowledge of which proteins are present. 

  Targeted quantification: analysis of a preselected group of proteins (hypothesis driven, need prior knowledge) 

• DDA: Quantification of many proteins (1000) in relaively few samples. Targeted: only 10-100 proteins can be identified.

• Targeted: More precise, accurate and reproducible quantification (higher sensitivity ad dynamic range) 

• DIA = Data independent aquisition: attempt to take the best of bot shotgun and targeted: measure verything (withput prior knowledge), the analyse selected anlytes (with prior knowledge) 

  • to quantify large numbers of proteins and peptides over many samples

  • when no need of maximum selectivity and sensitivity

  • when POI change in the future

TMT: Tandem mass tags. TMT labeling is a widely used method for quantitative proteomics that allows for the simultaneous measurement of multiple samples in a single mass spectrometry experiment. It involves the chemical labeling of peptide samples with isobaric tags, each of which contains a unique mass reporter ion that can be used to quantify the relative abundance of the labeled peptides across multiple samples.

Combining TMT labeling with DIA provides a powerful approach for high-throughput quantitative proteomics. TMT labeling allows for the multiplexed analysis of multiple samples in a single DIA experiment, enabling the quantification of thousands of proteins across multiple conditions or timepoints.

Furthermore, TMT labeling can reduce the complexity of the DIA data by pre-fractionating the samples prior to analysis, which reduces the number of peptides in each DIA window and simplifies the identification and quantification of the peptides.