質量分析（MS）を用いたタンパク質の定量に

 

 

 

 

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QPrEST^TM Mass Spectrometry Standards

 

Introducing QPrEST, a new standard suitable for mass spectrometry (MS)-based quantification. The QPrEST product line represents a new category of isotope-labeled internal MS standards offering distinct advantages to existing products for relative and absolute quantification. QPrESTs are protein fragments containing a 50-150 amino acid sequence identical to part of a human protein target.

 

 

The New Standard for Quantitative Proteomics

 

Added early in the proteomics workflow Atlas Antibodies QPrEST products generate multiple labeled peptides serving as internal standards for absolute quantification of the corresponding endogenous proteins. Originally designed by the Swedish Human Protein Atlas project the QPrEST standards are produced on demand through metabolic labeling using heavy isotope labeled (¹⁵N, ¹³C) Lysine and Arginine residues resulting in more than 99% isotope incorporation. The QPrEST concentration is determined with high accuracy and precision by MS-analysis through the use of a proprietary N-terminal Quantification Tag (QTag) sequence present in all QPrESTs.

 

 

QPrESTs in Scientific Research

 

The QPrEST concept has until now been used for single- and multiplex determination of protein copy numbers in HeLa cells and for mapping the murine platelet proteome. Also, the correlation between gene transcript levels and protein content have been investigated using a combination of RNA sequencing and QPrEST methodologies. The QPrEST concept enables accurate and streamlined quantification of the absolute or relative amount of proteins in a wide range of proteomics applications.

 

• Zeiler M et al. A Protein Epitope Signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines. Mol Cell Proteomics 2012 Mar 11;3;O111.009613

 

• Edfors F et al. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. Mol Cell Proteomics 2014 Jun 13;6; 1611-24

 

Zeiler M et al. Copy number analysis of the murine platelet proteome spanning the complete abundance range. Mol Cell Proteomics 2014 Dec 13;12; 3435-45

 

 

 

 

Extensive Proteome Coverage

 

Atlas Antibodies QPrEST line currently includes 19,000 products corresponding to >13,000 unique human targets, available to you as absolute quantification standards. All QPrESTs are produced and quality controlled on demand from our in house Human Protein Atlas derived clone collection with an estimated delivery time of three weeks. The products are covered by Atlas Antibodies Guarantee Program and our experienced and dedicated support team are waiting to assist you with order and product related questions.

 

 

 

Comparison of QPrEST^TM concept and other types of internal standards

 

Product	QPrEST	AQUA Peptide	Full-length Protein
Number of Peptides	Multiple	Single	Multiple
Spike-in	Early	Late	Early
Isotopic Enrichment	>99%	>99%	>90%
Purity	>80%	>95%	>80%
Concentration Accuracy	(High) ESI-MS/MS	(High) Amino acid analysis	(Low) BCA
Concentration Precision	+/- 10%	+/- 5-25%	N/A
Modifications	No	Optional	Depending on system
Listed Targets	>13,000	>1,000	> 9,000

 

 

 

Multiple Peptides in a Single QPrEST

 

Tired of always testing multiple peptide standards in your MS-setup? With the QPrEST technology you can improve your proteomics workflow by generating quantitative data from more than one peptide using a single isotope-labeled standard leading to better data consistency. All QPrESTs contain a minimum of two (average 3.2) unique, tryptic peptides matching your target protein sequence.

 

 

The QPrEST includes a sequence of 50-150 amino acids, with at least two tryptic peptides identical to a human protein target sequence.

 

 

 

Better workflow with early spike-in of QPrEST

 

Extensive sample preparation can affect the accuracy of your quantitative data. As the QPrEST is added to the protein sample prior to trypsin digestion, the results will be minimally affected by losses during sample preparation, providing you with highly accurate quantitative information based on multiple tryptic peptides. The presence of the QPrEST standard during trypsin digestion also reduces the potential bias resulting from incomplete cleavage by the proteolytic enzyme, a problematic issue for peptide standards.

 

 

The QPrEST can be added to the sample at an early stage in the proteomics workflow, decreasing the bias introduced during sample preparation.

 

 

 

More than 99% Incorporation Efficiency of Isotope Labeled Amino Acids

 

Using a special Escherichia coli  BL21(DE3) derivative auxotrophic for Lysine and Arginine the incorporation of ¹³C and ¹⁵N isotope labeled amino acids is comparable to that obtained for synthetically produced peptides and superior to available full-length recombinant protein standards.

 

 

QPrESTs are produced in an E. coli strain auxotrophic for Lysine and Arginine. The addition of (1) heavy isotope-labeled versions of Lysine and Arginine and (2) unlabeled versions of the remaining 18 amino acids results in a heavy isotope-labeled QPrEST.

 

 

 

High accuracy and precision in QPrEST concentration determination

 

All QPrESTs contain a proprietary Quantification Tag (QTag) sequence that has been used for accurate and precise determination of the QPrEST concentration. For this purpose, we have used an unlabeled, ultra pure and amino acid analyzed QTag standard as the internal reference. All QPrESTs have been quantified through MS-analysis of multiple QTag peptides resulting in measurements of high accuracy and precision matching those obtained from the more laborious and expensive amino acid analysis. We provide our customers with a ready-to-use isotope-labeled internal standard quantified with a precision of +/- 10%. Importantly, since QPrEST quantification is performed against the unique QTag sequence only, contaminating molecules will not affect the concentration determination and complete QPrEST purity is not essential.

 

 

The QPrEST concentration is determined using an MS-based setup including the QTag standard.

 

 

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