**4.1 Cells**

436 Modern Metrology Concerns

3. LC/MS: After proteolysis by trypsin, the sample containing the internal standards was subjected to LC/MS. The amounts of unlabeled and labeled peptides were monitored by revered-phase LC/ESI-quadrupole MS using certain ion traces. Comparing the signal ratio of unlabeled and labeled peptides for the standard solution of known concentration to that for the unknown sample, the quantity of hGH was calculated.

Very recently, isotope-labeled intact proteins have been successfully used for MS based protein quantification (Brun et al., 2007; Janecki et al., 2007; Peng et al., 2004). This approach is based on the use of isotope labeled full-length proteins produced by recombinant DNA techniques as internal standards for quantification. The advantage of this approach is that it is unnecessary to take into account the efficiency of hydrolysis/proteolysis that is the main limitation of amino acid or peptide analysis approaches. As the target analyte and the internal standard are chemically identical, they act exactly the same way during not only pre-analytical treatments such as proteolysis, but also LC/MS measurement. For this reason, this analysis can provide more accurate quantitative analysis than the other MS-based approaches described above, minimizing the influences of experimental variations. However, the difficulty of preparation of isotopically labeled protein has been an obstacle. The following is one representative example of how to quantify a certain protein in a complex biological sample using an isotope-labeled intact protein. Janecki et al accurately measured the level of the human alcohol dehydrogenase (ADH1C1) in liver extracts using

1. Preparation of the stable isotope-labeled ADH1C1: Labeled ADH1C1 was expressed in *Eschericha coli* that were cultured in specific media containing isotopically labeled Lys. The protein was purified by affinity chromatography using His-tags at the N-terminus. The purity and activity of purified ADH1C1 were verified by sodium dodecyl sulfate

2. Sample preparation: The labeled standard protein was added to liver samples in two defferent ways. One set referred to as experiment 1 contains various concentrations of the standard in a constant amount of liver extracts. The other set referred to as experiment 2 has a constant amount of the labeled protein in various concentrations of liver extracts. Both experiments were applied to quantification of ADH1C1 in liver extracts to generate statistically significant quantitative data. The mixture was then

3. Quantitative analysis by HPLC-MS/MS: Prior to the actual analysis, preliminary experiments were performed using unlabeled ADH1C1 to optimize the multiple reaction monitoring (MRM) transitions , and a specific target peptide was selected. And then, the retrieved peptides from proteolysis were subjected to HPLC-MS/MS using MRM mode. For quantitative purpose, analyst quantification module software was

Determination of the exact amounts of proteins gives valuable information about biological mechanisms of targeted proteins and becomes crucial for the evaluation of various biomarker candidates. Until recently, immunoassays such as ELISA were considered as the

used to calculate the target peaks generated from heavy and normal peptides.

followed by trypsin digestion, and resulting peptides were recovered.

**3.3.3 Analysis using isotope labeled intact proteins** 

in-vitro expressed labeled ADH1C1 (Janecki et al., 2007).

(SDS)-PAGE and a specific activity assay.

**3.4 Summary** 

A cell is the functional basic unit of life and is much larger and complex than nucleic acids or proteins. Therefore, metrology for cells will be of substantially greater degree of complexity. Recent breathtaking advances in the attempt of utilization of cells in regenerative medicine (Mimeault et al., 2007) draw great attentions of metrologists in biology. Although unconceivable degree of technical difficulties are expected, metrology for cells need to be established to support the advances in regenerative medicine to turn into safe and effective means to restore health of many impaired or elder people. In fact, safety and efficacy of regenerative medicine are debated subjects at present (Ilic and Polak, 2011). In addition, many animal tests are being replaced by cell-based tests to avoid ethical problems, and the validity of cell-based testing will require rigorous examination of cells used for such tests to result in harmonious and valid outcomes. International cell depositories such as American Types of Cell Cultures (ATCC) are the major bodies performing R&D for collective testing of cells, which is necessary for QC and QA of reserved cell resources. In dealing with cells, authentication of the target cells is a key issue, which should be confirmed with effective and valid tests. For example, one of the ATTC R&D programs, Developmental Biology Program (http://www.atcc.org/Science/ CollectionsResearchandDevelopment/DevelopmentalBiologyandStemCells/tabid/207/Def ault.aspx), employs state-of-the-art technologies for the authentication of human ES (embryonic stem) cell lines, mouse ES cell lines and mouse feeder cells. Five critical tests address the major concerns of researchers and clinicians working with human ES cell lines and are conducted routinely by ATCC:


Appropriateness of such tests is to be determined by experts in the field, and the quantities here are more likely method-dependent. As discussed in the later section, metrology for such quantities needs to be established to support 'metrological execution' of the given measurement protocols, where those protocols is not fully established yet. In this regard, the advancement of cell testing protocols should be closely tracked for prompt implementation of metrology in such testing protocols.

Counting of well specified cells is rather straight forward in terms of quantification. CCQM BAWG has executed or is preparing for two international comparisons: 1) flow cytometric determination (CCQM-P102) and 2) microscopic determination (CCQM-P123). It is noteworthy that physical parameters such as motility and morphology could be important in characterization of cells. Among NMIs, NIST scientists are in particular active in such investigations (Ni and Chiang, 2007).
