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SILu™PrEST – Isotope-labeled Multipeptide Standards for Quantitative Mass Spectrometry

Introduction

Within the field of proteomics, mass spectrometry (MS) has become the method of choice for protein analysis.1 Using MS, thousands of proteins from a complex sample can be identified in a single run. Apart from enabling protein identification, MS has also become a key tool for protein quantification. One strategy to obtain highly accurate measures of protein abundance within a sample is to use a heavy isotope-labeled standard, preferably spiked into the sample at an early stage of the sample preparation procedure. In order to obtain reliable quantitative data, it is desirable to quantify multiple peptides for each target protein. Several types of isotope-labeled standards exist, one being Atlas Antibodies’ quantitative standard SILuPrEST. This standard possesses several beneficial properties such as high incorporation efficiency of heavy isotope-labeled amino acid residues. It also contains multiple unique tryptic peptides, increasing the reliability of the quantitative measurement of the corresponding endogenous target protein.

SILuPrEST Isotope-Labeled Standards

The SILuPrEST standards were originally derived from the Human Protein Atlas project,2,3 where unlabeled (light) recombinant protein fragments are used as antigens for antibody generation. These protein epitope signature tags (PrESTs) can be produced with incorporated heavy isotope-labeled amino acids to generate proteins with unique properties that make them suitable as internal standards for mass spectrometry-based quantification.4-6 All SILuPrESTs contain a stretch of 50–150 amino acids identical to a target human protein sequence including at least two unique tryptic peptides. The SILuPrEST standard can be added to the sample at an early stage of the sample preparation workflow, decreasing the variation introduced during proteolytic cleavage. An N-terminal quantification tag (QTag) is present in all SILuPrEST standards, and is used for accurate quantification of the SILuPrEST using an unlabeled QTag protein as internal reference (Figure 1). The QTag consists of a hexahistidine tag genetically fused to an albumin binding protein (ABP) sequence derived from streptococcal protein G.Quantification of SILuPrEST standards is based on quantitative analysis of multiple tryptic peptides spanning the QTag sequence.

Quantification of Endogenous Protein using SILu™ PrEST

Figure 1. Schematic figure of a SILuPrEST standard (gray). The N-terminal part of the sequence consists of the QTag sequence, used for purification and accurate quantification of the SILuPrEST using an unlabeled QTag (red). The C-terminal part of the sequence is identical to a portion of a human protein (blue). This part is used for absolute quantification of the endogenous target protein.

 

Proteome Coverage

The SILuPrEST product catalog contains over 20,000 products, of which over 70% have at least one experimentally verified proteotypic peptide, based on data from PeptideAtlas7 (Figure 2). In total, the currently available SILuPrEST standards target more than 13,000 human proteins, of which more than 40% are covered by multiple (up to five) SILuPrEST standards.

Number of Unique Tryptic Peptides

Figure 2. Distribution of SILuPrEST products based on number of unique tryptic peptides (red bars). The number of SILuPrESTs within each group with at least one tryptic peptide present in PeptideAtlas is also presented (gray bars).

 

Production and Quality Control

Heavy isotope-labeled SILuPrESTs are expressed in an Escherichia coli BL21(DE3) derivative strain, auxotrophic for lysine and arginine.8 The cell cultivation is performed in a minimal auto induction medium and isotopic incorporation is achieved by the addition of heavy isotopelabeled arginine and lysine (13C, 15N) to the culture. The QTag part of the protein sequence contains an N-terminal hexahistidine tag used for affinity purification of the SILuPrEST standard. After purification, SILuPrEST purity is verified using SDS-PAGE and the correct protein molecular weight is confirmed using ESI-MS analysis (Figure 3).

The auxotrophic E. coli strain contains deletions in the lysA and argA genes making the cells unable to survive without the addition of arginine and lysine to the growth medium (Figure 4A). The auxotrophy results in a near complete (>99%) isotopic incorporation as verified through the absence of peaks corresponding to unlabeled peptides in an MS spectrum (Figure 4B).

The SILuPrEST standards are stable and no effect on the SILuPrEST concentration has been observed after repeated freeze-thaw cycles.

Quality control of SILu PrEST standard

Figure 3. Quality control of SILuPrEST standard. (A) SDS-PAGE is used to determine protein purity and (B) the protein molecular mass is verified with ESI-MS.

 

Verification of isotopic incorporation

Figure 4. Verification of isotopic incorporation. (A) The Lys and Arg auxotrophic E. coli BL21(DE3) strain is dependent on addition of Arg and Lys when grown on minimal medium. (B) Analysis of SILuPrEST tryptic digests using ESI-MS shows no peaks corresponding to unlabeled peptides can be detected.

 

Application Example of SILuPrESTs as Internal Standards

Protein quantification using SILuPrEST standards can be performed either in single-plex, or in a multi-plex format where a set of SILuPrEST standards is added to the sample for parallel analysis of multiple target proteins. SILuPrEST standards have been used as internal references to determine the copy number of proteins in cell lines.4,5 A set of SILuPrEST standards was spiked into a HeLa cell lysate directly after cell lysis and the sample was digested using the filter-aided sample preparation (FASP) methodology. Generated peptides were further fractionated into six samples using strong anion exchange chromatography in a pipette tip format. Peptide fractions were desalted and separated using a 3 h LC gradient prior to injection on a QExactive mass spectrometer. Figure 5 shows two examples of protein quantification using SILuPrEST standards.4 The protein UGDH was quantified using a total of five peptides generated from two separate SILuPrEST standards, resulting in a determined copy number of 890,000 copies per cell. CAPG was quantified using one SILuPrEST and a total of four peptides. The copy number was for this protein determined to be 2.5 million copies per cell.

Absolute quantification of two human proteins using SILu PrEST standards

Figure 5. Absolute quantification of two human proteins using SILuPrEST standards. The copy numbers of CAPG and UGDH were determined in HeLa cells. Quantified peptides are shown on the x axis and determined copy numbers on the y axis. Each circle represents data from one of three replicate analyses.

 

Accurate SILuPrEST Quantification

Labeled SILuPrEST standards are accurately quantified using the QTag part of the protein. An unlabeled version of the QTag is used as an internal reference in an MS-based setup where multiple QTag-derived peptides are used to quantify the SILuPrEST standard.5 A high purity of the QTag is ensured through multiple affinity purification steps based on both N- and C-terminal purification handles, and the QTag concentration is accurately quantified using amino acid analysis. The heavy isotopelabeled SILuPrEST is mixed with the unlabeled QTag protein and the sample is digested. Peptides are further analyzed using an LC-ESI-QTOF setup where ratios between light and heavy QTag-derived peptides are used to determine the absolute SILuPrEST concentration (Figure 6). The analysis is not limited to fully cleaved tryptic peptides, as the equal digestion efficiency between SILuPrEST and QTag results in very similar peptide ratios for fully cleaved tryptic peptides and peptides containing one or two intact tryptic cleavage sites. Accurate quantification of the endogenous target protein depends on the quantitative precision of the added standard. Based on data from three replicate experiments, the concentration is determined with an imprecision below 10%.

SILu™ PrEST quantification using three example QTag peptides

Figure 6. SILu™PrEST quantification using three example QTag peptides. Extracted ion chromatograms are shown to the left. Extracted spectra with peaks corresponding to both labeled and unlabelled peptides are shown to the right. The determined L/H ratio for each peptide is also presented. The three peptides are marked in the QTag sequence at the bottom of the figure.

 

Summary

  • SILuPrEST are recombinantly produced, heavy isotope-labeled, protein standards for MS-based absolute quantification
  • The product catalog contains over 20,000 bioinformatically selected SILuPrEST standards with proteome-wide coverage
  • An accurate MS-based setup with an unlabeled internal QTag reference standard is used for precise SILuPrEST quantification to ensure reliable downstream results
  • SILuPrEST standards have been used to determine absolute protein copy numbers in different cell lines

 

References

  1. Aebersold R and Mann M. (2003) Mass spectrometry-based proteomics. Nature 422, 198–207
  2. Uhlén M et al. (2015) Proteomics. Tissue-based map of the human proteome. Science 23;347(6220).
  3. Uhlén M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, Zwahlén M, Kampf C, Wester K, Hober S, Wernérus H, Björling L and Pontén F. (2010) Towards a knowledge-based Human Protein Atlas. Nat Biotechnol 28, 1248–1250.
  4. Edfors F, Boström T, Forsström B, Zeiler M, Johansson H, Lundberg E, Hober S, Lehtiö J, Mann M and Uhlén M. (2014) Immuno-proteomics using polyclonal antibodies and stable isotope labeled affinity-purified recombinant proteins. Mol Cell Proteomics 13, 1611–1624.
  5. Zeiler M, Straube WL, Lundberg E, Uhlén M and Mann M. (2012) 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 11, O111 009613.
  6. Zeiler M, Moser M and Mann M. (2014) Copy number analysis of the murine platelet proteome spanning the complete abundance range. Mol Cell Proteomics 13, 3435–3445.
  7. Farrah T, Deutsch EW, Omenn GS, Sun Z, Watts JD, Yamamoto T, Shteynberg D, Harris MM and Moritz RL. (2014) State of the human proteome in 2013 as viewed through PeptideAtlas: comparing the kidney, urine, and plasma proteomes for the biology - and disease-driven Human Proteome Project. J Proteome Res 13, 60–75.
  8. Matic I, Jaffray EG, Oxenham SK, Groves MJ, Barratt CL, Tauro S, Stanley-Wall NR and Hay R. T. (2011) Absolute SILAC-compatible expression strain allows Sumo-2 copy number determination in clinical samples. J Proteome Res 10, 4869–4875.