Introduction of mass spectrometry and sample preparation

Proteomics

• Proteomics is the large-scale study of proteins which are vital parts of living organisms, with many functions.
• The proteome is the entire set of proteins produced or modified by an organism or system.
• Proteomics enables the identification of ever-increasing numbers of proteins with time and distinct requirements, or stresses, that a cell or organism undergoes.

Proteomics can reveal phenotype

• Currently, NGS is more commonly used in clinical application than proteomics.
• Genomics cannot promise the detected mutations can really influence the cell regulation.
• Although transcriptomics can measure the gene expression, the correlation of expression values between proteins and RNAs are not high.
• Most of the regulators are proteins, proteomics can be used to understand regulation directly.

Silence mutation

• Silence mutation
• Change a codon into a mutant codon that specifies exactly the same amino acid.
• The majority of silent mutations change the third nucleotide of a codon.

Conservative missense mutation

• Conservative missense mutation
• The substituted amino acid has chemical properties similar to the one it replaces, and then it may have little or no effect on protein function.

​

Mass spectrometry based proteomics

Mass spectrometry

• Mass spectrometry is a sensitive analytical technique used to detect, identify and quantitate the analytes in a sample based on the mass-to-charge (m/z) ratio of the analyte ions in the gas phase.

​

Sample

HPLC

Mass-Spectrometer

Mass spectrometry

High Vacuum

Mass Analyzer

Mass spectrometer

Quadrupole mass analyzer

• Only ions with a specific mass-to-charge ratio (m/Q) reach the detector. A spectrum is recorded by scanning through measurement of each m/Q of interest.

Time-of-Flight Mass Analyzer

• The measurement is similar to a race: a group of ions is accelerated by an extractor (start of the race), which causes them to drift through the flight tube (the race course) toward a detector (the finish line).
• A molecule with a large mass should travel more slowly than a molecule with a small mass, so, the elapsed time indicates the molecular mass when ions have identical charge.

MS-based proteomics approaches

Ion

Source

High Vacuum

Mass

Analyzer

MS₁

MS₂

Mass

Analyzer

X

X

X

X

X

X

X

X

Proteins

Peptides

Digestion

Top-Down

Bottom-Up

==

Shotgun

MS-based proteomics approaches

m/z

MS₁

MS/MS

MS/MS

m/z

m/z

MS1 and MS2

• In MS1 level, the peptides are detected by their intensity. Which amino acids are involved in the peptides still unknown. Thus, MS1 is normally for quantification.
• In MS2 level, the peptides are cleaved to small pieces which m/z can be known and refer to the database. Thus, the amino acid composition of the peptides can be understanded. MS2 is for peptide identification.

Feature detection

Peptide isotope patterns in the MS spectra

1 Peptide != 1 Peak

Feature detection – 3D peak detection

• 2D peaks are assembled into 3D peaks
• Two 2D peaks in adjacent scans are connected when Δm < 7ppm
• Also next to nearest scan is checked

3D

2D

3D

Feature detection – 3D peak detection

• 2D peaks are assembled into 3D peaks
• Two 2D peaks in adjacent scans are connected when Δm < 7ppm
• Also next to nearest scan is checked

3D

2D

3D

Feature detection – Isotope pattern assembly

3 main criteria

• Mass difference ̴ 1/z

​

• Retention time dependence of intensity

​

• Relative intensities in isotope pattern

Feature detection – Individual peptide mass tolerances

For each peptide mass calculate:

• mean
• standard deviation

Peptide isotope patterns in the MS spectra

1 Peptide != 1 Peak

Intensity

m/z

Peptide with X Carbons

X ¹²C

(X-1) ¹²C

1 ¹³C

(X-2) ¹²C

2 ¹³C

(X-3) ¹²C

3 ¹³C

1

1

1

Peptide isotope patterns in the MS spectra

Intensity

m/z

Intensity

m/z

1

1

1

0.5

0.5

0.5

Intensity

m/z

0.333

0.333

0.333

Charge +1

Charge +2

Charge +3

Peptide isotope patterns in the MS spectra

Intensity

m/z

999

1000

1001

1002

Intensity

m/z

1

1

1

0.5

0.5

0.5

500

500.5

501

501.5

Intensity

m/z

333.6

333.933

334.266

334.599

0.333

0.333

0.333

Charge +1

Charge +2

Charge +3

Feature detection

Peptide Fragmentation Nomenclature

a₁

c₁

x₂

z₂

a₂

c₂

x₁

z₁

Roepstorff P, Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984 Nov;11(11):601. PMID: 6525415.

b₁

y₂

b₂

y₁

Fragmentation Modes in Scaffold

• CID (Collision Induced Dissociation):
• Collision Induced Dissociation produces B ions and Y ions predominately (note Scaffold annotates ions as B or Y).

Fragmentation Modes in Scaffold

• HCD (Collision Induced Dissociation):
• Higher Energy Collision Induced Dissociation also produces B ions and Y ions predominately. (note Scaffold annotates ions as B or Y).

Fragmentation Modes in Scaffold

• ETD (Electron Transfer Dissociation):
• Electron Transfer Dissociation produces C ions and Z ions predominately (note Scaffold annotates ions as C or Z).

Fragmentation Modes in Scaffold

Fragmentation ions

CID/HCD

-17

-17

Mass spectrometry based proteomics

Experimental design

• A study is valuable may strongly rely on the experimental design.
• Several factors need to be considered
• Sample groups – how to define the control and experiment groups.
• Sample types – what kind of samples need to be used, bacterial, tissue, blood, etc.
• Techniques – label-free, isobaric labeling, fraction, reference, etc.
• A thorough discussion between biologists, physicians and bioinformaticians is very crucial.

Sample groups

• Defining sample groups needs to match the research topic.
• The key factor for comparison should cause a clear separation between control and experimental groups.
• The other factors should be as diverse as possible, and the variance between groups should be small.
• The common factors which need to be considered are age, gender, nation, health, etc.

Replicates

• Replicates are the repeated experiments. There are several reasons for performing replicates
• Avoid bias or special cases – a sample shows a special phenomena cannot represent the whole set. Ex: a student get A+ in a class, it does not all students get A+.
• Reproducibility – if a phenomena does exist, it should be reproduceable. The phenomena only shows one time does not mean it do exist, ex: contaminant.
• Statistical analysis – in order to make the results comparable, performing statistical analysis is required. Statistics need multiple samples for computing significance.

Biological replicate

• Biological replicate is repeating the same experiment by using different samples from a group.
• For example, selecting three colonies of E. coli which have a specific gene knockout. The colonies are different ones, but the experimental condition/group is the same.
• Biological replicate is mainly for avoiding the issue of special case in order to understand the general situation of the experimental conditions.

Technical replicate

• Technical replicate is performing the same experiment with identical samples.
• For example, selecting a colony of E. coli which have a specific gene knockout. Using the same colony to perform the experiment multiple times.
• The purpose of setting technical replicate is for minimizing the influences caused by technical issue. Or the samples which cannot have biological replicates.
• In general, biological replicates are more important than technical replicates

Fraction

• Since proteins have no ability to amplify or reproduce themselves, the only way to increase the depth of sample pool is using fraction.
• Fractionation refers to the separation of proteins into smaller groups or fractions before mass spectrometry analysis.
• Reduce sample complexity
• Increase the depth of protein coverage
• Improve the detection of low-abundance proteins.
• Fractionation can be achieved using a variety of techniques
• Gel-based fractionation
• Liquid chromatography
• Isoelectric focusing.
• Normally fractions of the same sample group are merged during analysis.