Application Note 40

Full-Length Expressed Stable Isotope-Labeled Proteins for Quantification

Waltraud Mair,¹ Sasha Singh,^1,2,3 Judith Steen¹ and Hanno Steen²

1. F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Boston, MA USA
2. Department of Pathology, Boston Children’s Hospital, Boston, MA USA
3. Center for Interdisciplinary Cardiovascular Sciences, Brigham and Women’s Hospital, Boston, MA USA

FLEXIQuant, short for Full-Length Expressed Stable Isotope-Labeled Proteins for Quantification, utilizes stable isotope-labeled (“heavy”) full-length recombinant proteins which are expressed in vitro using WEPRO^® and other reagents from CellFree Sciences (CFS). The detailed workflow of FLEXIQuant is depicted in Figure 1, with more details published elsewhere.^1,2 The isotopic labeling of the heavy protein standards is achieved by incorporating ¹⁵N- and/or ¹³C-labeled amino acids, preferably lysine and arginine. Prior to trypsinization, similar amounts of the heavy protein standard and the unlabeled endogenous sample protein are combined. The unlabeled (“light”) protein of interest and the heavy protein standard can be purified separately beforehand. Alternatively, the WEPRO extract with the recombinant heavy protein standard can be mixed with the cell or tissue lysate containing the light endogenous protein of interest prior to simultaneously purifying by immunoprecipitation the light and the heavy proteins. After trypsinization, the digest is analyzed by mass spectrometry (Figure 1 and 2). All unmodified peptides will be present as pairs featuring the light and the heavy isotopologue, whereby the light-to-heavy (L/H) intensity ratio of these peak pairs reflects the initial mixing ratio. Any deviation from this ratio indicates a) that a fraction of the endogenous peptides was modified and thus shifted in the mass-to-charge (m/z) ratio and the retention time domain, and b) the extent of this modification: if the endogenous peptide is present only at 20% of the intensity expected based on the ratio of all other modified peptides, it can be assumed that 80% of this peptide was modified.

An additional feature of the FLEXIQuant approach is the presence of a unique peptide, the FLEX peptide, next to the His6-tag used for purification. This FLEX peptide can be used to determine the absolute quantification of the heavy recombinant protein using “inverse” isotope dilution mass spectrometry – “inverse” as the synthetic peptide used for quantification is not labeled because the analyte of interest, i.e. the full-length recombinant protein, is labeled. Once the absolute concentration of the heavy recombinant protein is determined, the absolute concentration of the endogenous light protein can be inferred.

An example application of FLEXIQuant is shown in Figure 2. In this particular example, HeLa S3 cells were arrested in prometaphase using nocodazole.¹ Aliquots were removed four, eight and 12 hours after initiating the nocodazole treatment. After cell lysis in the presence of phosphatase inhibitors, wheat germ extract containing heavy CDC27 was added to the lysate. The light endogenous and the heavy exogenous CDC27 were co-purified by immuno­precipitation. After trypsinization, the digest was analyzed by liquid chromatography mass spectrometry (LC-MS). All observable CDC27-derived peptides presented themselves as isotopologue pairs. Those peptides that became modified during the incubation with nocodazole showed a decrease in the L/H ratio (see Figure 2A and B), which i) identifies the modified region of CDC27 during mitotic arrest, ii) provides information about the extent of modification, and iii) provides insights into the modification kinetics. More detailed analysis identifies the modifications as phosphorylation (see Figure 2A, green arrow).

Additional examples for applications of the FLEXIQuant strategy and derivatives thereof, such as FLEXIQinase,⁵ (Figure 1) have been published.^3,4,5

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