Designer proteins comprising concatenated tryptic peptides can be used for system calibrations (HPLC, MS, antibody/peptide arrays) and to explore the kinetics of proteolysis of cleavage sites in  different primary sequence contexts.

QconCATs introduce the concept of ‘designer proteins’ to proteomics. Specific QconCAT proteins can be used to assess the properties of defined peptides in HPLC, MS and MS/MS analysis. In addition, QconCATs can be used in the quality control of antibody or peptide arrays and to explore the kinetics of proteolysis of cleavage sites in different primary sequence contexts.

Calibration of HPLC separations and MS instruments

Calibration standards for MS instrumentation are typically commercially available peptides. PolyQuant has designed a QconCAT (QCAL) for use in the optimisation and standardisation of all commonly used instrumentation platforms for proteomics. QCAL is a QconCAT (52.2kDa), comprising 22 unique peptides, ranging from ~ 410-3100Da. QCAL was designed to provide standards for peptide separation by reversed-phase chromatography, to facilitate the assessment and optimisation of instrument resolution and to evaluate the linearity of signal detection in different MS instruments, including MALDI-ToF, ESI MS and FTICR (1). QCAL also comprises peptides containing amino acid residues subject to modification (oxidation of methionine, deamidation of gln/asn) and modification of lysine residues by guanidination (2), allowing the routine monitoring of these processes.

QCAL is readily digested to completion under standard tryptic digestion conditions, is readily produced in large amounts and can be made available for use in the proteomics community.

Exploring MS signal intensities

Designer proteins like QconCATs provide elegant tools to determine the primary sequence dependence of ionisation efficiency in a variety of MS platforms. Proteins can be designed to include a systematic series of amino acid sequences, which upon cleavage, yield a range of peptides that alter systematically, varying one feature at a time. For the cost of 5nmol of five synthetic peptides, μmol quantities of a QconCAT, encoding approximately 20 synthetic peptides, is a far more attractive proposition!

Quality control of antibody and peptide arrays

QconCATs, in unlabelled form could be used to standardise/QC the performance of peptide arrays, or anti-peptide antibody arrays, during development and as controls for generating standard curves during high throughput competition experiments. The 1:1:1 stoichiometry of peptides derived from digestion of a QconCAT protein would allow evaluation of the performance of each peptide in an array. In addition, a QconCAT in unlabelled form could be generated comprising a concatenation of difficult to synthesize/larger peptides that were refractory to chemical synthesis. After endopeptidase digestion an HPLC purification step would be required to separate the peptides and each peptide would then be quantified separately.

Exploring the kinetics of proteolysis

Designer proteins like QconCATs provide an elegant method to evaluate the primary sequence dependence of the kinetics of proteolysis. Proteins can be designed to include any amino acid sequence context around endopeptidase cleavage sites. The kinetics of cleavage of multiple contexts can be determined in the same experiment and accurately quantified by comparison with a completely digested, heavy isotope labelled version of the same QconCAT.

References

1) Eyers et al. (2008). QCAL–a novel standard for assessing instrument conditions for proteome analysis. J Am Soc Mass Spectrom 19(9):1275-80.

2) Brancia, F.L. et al. A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics. Electrophoresis 22, 552–559 (2001).