Proteomics

The proteome is the entire set of proteins expressed by a cell/organism at a certain time. The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations (Anderson and Anderson, Electrophoresis, 1998). Liquid chromatography mass spectrometry-based (LC-MS) proteomics allow a large-scale identification and quantification of proteins from complex samples. Several thousands of proteins can be analyzed in a single LC-MS run.

Q Exactive HF OrbiTrap

A general proteomic and phospho- proteomic profiling scheme, which we use in our lab is shown in Figure 1. About 100 ug of total protein is necessary to detect about 6.000 proteins. For a comprehensive phospho-proteome, 15 mg total protein is necessary to identify about 12.000 unique phosphorylated peptides in a mammalian proteome. Phosphorylated peptides have to be enriched prior analysis.

Figure 1

SCX strong cation exchange<br />SAX strong anion exchange<br />TiO<sub>2</sub> Titaniumoxide Zoom Image
SCX strong cation exchange
SAX strong anion exchange
TiO2 Titaniumoxide

Here, you see MS OrbiTrap spectra of two different peptides. Multiple peaks per peptide are caused by 13C6 isotopes. How can you calculate the molecular weight (MW) of a peptide?

Figure 2

H = mass of hydrogen<br />n = charge Zoom Image
H = mass of hydrogen
n = charge

 

Peptides need to be further fragmented in order to read the sequence. There are different fragmentation methods available, resulting in different fragmentation patterns. The Q Exactive HF is applaying Higher-energy C-trap dissociation (HCD), specific y and b ions are generated as shown in Figure 3.

Figure 3

<p>Peptide fragment ions are indicated by b, if the charge is retained on the N-terminus and by y, if the charge is maintained on the C-terminus</p> Zoom Image

Peptide fragment ions are indicated by b, if the charge is retained on the N-terminus and by y, if the charge is maintained on the C-terminus

QTrap 6500, targeted MRM analysis

For the QTrap 6500, peptide specific methods have to be established and optimized. At least three transitions per peptide are necessary for identification, they can be quantitated and compared to other samples. Figure 4 shows several transitions from our BSA standard run over 75 minutes, Figure 5 displays three MRM transition from one specific peptide in detail and its according MS2 spectrum.

Figure 4

 

 

Figure 5

Three transitions (MRM's) of the tryptic peptide LVNELTEFAK are shown on the left, its entire MS2 spectrum on the right side. Zoom Image
Three transitions (MRM's) of the tryptic peptide LVNELTEFAK are shown on the left, its entire MS2 spectrum on the right side. [less]
 
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