International standard protein quantitation

Liu et al. (2003) focused their method paper on the instrumental set-up for on-line capillary LC coupled with MS-MS via a low flow-rate interface, hoping that miniaturization would improve sensitivity for many biomedical applications. The system design and perfonnance were tested on DHEA sulfate and pregnenolone sulfate quantitation from 5 pL of plasma from one male volunteer. Sample preparation was simple - addition of aqueous methanol and of the deuterated analog internal standards, protein precipitation, supernatant filtration through a Cig bed, evaporation and reconstitution in 100 pL of mobile phase, of which 20 pL were injected for LC-MS-MS analysis. The DHEA sulfate concentration found was in agreement with literature values based on GC-MS and LC-MS studies. 

Figure 6.5 Representative two-dimensional mass spectrometric analyses of sphingomyelin molecular species in the alkaline-treated lipid extracts of mouse spinal cord in the positive-ion mode in the presence of LiOH. Lipid extracts from spinal cord of mice at 48 days of age were prepared by using a Bligh-Dyer extraction procedure as previously described [27]. A part of each lipid extract was treated with lithium methoxide as described previously [28] and the residual lipid extract was reconstituted with 50 pL of 1 1 CHClj/MeOH per milligram of original tissue protein. Each of the reconstituted lipid extracts was diluted 10 times prior to addition of a small amount of LiOH (10 pmol LiOH/pL). Positive-ion ESI mass spectrum in the full MS scan mode was acquired in the mass range from m/z 600 to 900 from the diluted lipid extract of mouse spinal cord, which displayed very low abundance ions corresponding to lithiated SM species. Neutral loss scans (NLS) as indicated were also acquired from the diluted lipid extract in the mass range. All scans were displayed after normalization to the base peak in individual scan. The ion at m/z 653.6 corresponds to the selected internal standard for quantitative analysis of SM species.

The feasibility of this approach to not only differentiate pathogenic and nonpathogenic strains of bacteria based on significant differences in protein mass but also on the basis of variations in levels of protein expression was demonstrated using a method for quantitating protein expression by LC/MS of whole proteins.54 This method is based on the fact that some proteins present in cells are abundant universal proteins whose expression levels exhibit little variation. This method demonstrates that these co-extracted proteins can be used as internal standards to which the other proteins in the sample can be compared. By comparing the intensities of a selected protein to a marker protein, or internal standard, a relative ratio is obtained. This ratio... 

Williams, T. L. Cahahan, J. H. Monday, S. R. Feng, P. C. Musser, S. M. Relative quantitation of intact proteins of bacterial ceh extracts using coextracted proteins as internal standards. Anal. Chem. 2004, 76,1002-1007. 

Both absolute quantitation and relative quantitation of species in mixtures is of interest in some circumstances. Quantitation in a 5-minute analysis can be achieved by addition of an internal standard, ideally the target microorganism grown in special media to incorporate heavy isotopes92-95 and determination of the relative peak heights of pairs of proteins from the analyte and the standard. Isotope-labeled proteins or peptides, selected to match proteins or peptides characteristic of target microorganisms, can also serve as internal standards for isotope ratio measurement. The addition of unmatched proteins or peptides is less reliable for either ESI or MALDI measurements because of unpredictable suppression in the variable mixture. 

As described above all samples were separated online using LCT ESI-TOF-MS then normalized for relative quantitation using a bovine insulin internal standard. Fractions were then collected for MAFDI-TOF-MS PMF, digested with modified porcine trypsin, and analyzed using the TofSpec2E. Following this analysis, three major classes of differentially expressed including proteins were revealed in these... 

O. A. Mirgorodskaya, Y. P. Kozmin, M. I. Titov, R. Kbmer, C. P. Sonksen, and P. Roepstorff. Quantitation of Peptides and Proteins by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Using lsO-Labeled Internal Standards. Rapid Commun. Mass Spectrom., 14(2000) 1226-1232. 

At an early stage in product development, there must be some material made available for use as ELISA reagents. ELISA requires a standard for quantitation and specific antibodies to the drug protein of interest. If these are not already available, either commercially or internally, then time, effort, and expense must be reserved for reagent development as well as assay development activities. The specific antibody is the key reagent in an ELISA. The antibody defines the specificity and sensitivity of the assay. To date there is no successful substitution for the routine production of specific antibodies by immunization of an animal with the antigen target of interest. 

Upon use of structurally modified variants as internal standards for the particular analytes, the relative quantificahon of oligonucleotides, peptides, and small proteins was demonstrated [44]. The potential of the ILM to allow quantitative analyses of peptides without the use of internal standards was presented recently [43]. Linear correlahons between peptide amount and signal intensities could be found upon applicahon of increased matrix-to-analyte ratios between 25,000 and 250,000 (mokmol). The dynamic range of linearity thus spanned one order of magnitude. Unfortunately, the importance of the M/A ratio prevents the use of this method in samples with unknown orders of concentration, for example, in a proteomics environment. On the other hand, the method is applicable for the screening of enzyme-catalyzed reactions because the starting concentrahons of the peptides are generally known in such assays. 

Determinative and confirmatory methods of analysis for PIR residue in bovine milk and liver have been developed, based on HPLC-TS-MS (209). Milk sample preparation consisted of precipitating the milk proteins with acidified MeCN followed by partitioning with a mixture of -butylchloride and hexane, LLE of PIR from aqueous phase into methylene chloride, and SPE cleanup. The dry residue after methylene chloride extraction was dissolved in ammonium hydroxide, and this basic solution was transferred to the top of Cl8 SPE column. The PIR elution was accomplished with TEA in MeOH. For liver, the samples were extracted with trifluoroacetic acid (TFA) in MeCN. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, and then extracted with methylene chloride. The organic solvent was evaporated to dryness, and the residue was dissolved in ammonium acetate. The overall recovery of PIR in milk was 94.5%, RSD of 8.7%, for liver 97.6%, RSD of 5.1 %. A chromatographically resolved stereoisomer of PIR with TS-MS response characteristics identical to PIR was used as an internal standard for the quantitative analysis of the ratio of peak areas of PIR and internal standard in the pro-tonated molecular-ion chromatogram at m/z 411.2. The mass spectrometer was set for an 8 min SIM-MS acquisition. Six samples can be processed and analyzed in approximately 3 hours. 

Macek et al. [120] developed a method to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The method is based on the protein precipitation with acetonitrile and a reversed-phase liquid chromatography performed on an octadecylsilica column (55 x 2 mm, 3 /im). The mobile phase consisted of methanol-10 mM ammonium acetate (60 40). Omeprazole and the internal standard, flunitra-zepam, elute at 0.80 0.1 min with a total rim time 1.35 min. Quantification was through positive-ion made and selected reaction monitoring mode at m/z 346.1 —> 197.9 for omeprazole and m/z 314 —> 268 for flunitrazepam, respectively. The lower limit of quantification was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. The method was applied to the analysis of samples from a pharmacokinetic study. 

Fig. 1. Purified proteins imaged using (A) Agilent 2100 Bioanalyzer and (B) traditional sodium dodecyl sulfide-polyacrylamide gel electrophoresis. (A) Note that the lowest two bands and the uppermost band are internal standards for sizing and quantitation. (B) 3-15% polyacrylamide gel stained with GelCode Blue. In both panels, the predicted size of the protein is labeled at the top of each lane.

In the recent method of Zienmiak, et al3 3, a liquid chranatographic procedure has been developed which separates and permits quantitative analysis of cimetidine (I), cimetidine sulfoxide (II), its hydroxymethyl (111) and guanol urea derivatives (IV). Metiamide, SK F 92058, is used as the internal standard. Their procedure involves the precipitation of protein with acetonitrile, addition of anhydrous K2HPOit, extraction of the separated aqueous phase with methylene chloride and KH2POi, to saturate and salt out the solution. The methylene chloride is evaporated to dryness, the sample is reconstituted with mobile phase (CHaCbhCE OIbl OtNihOH,... 

The following table provides a list of proteins that may be used as internal standards, along with their isoelectric points, pi, in quantitative applications of polyacrylamide gel electrophoresis. These proteins may be used in isoelectric focusing or in SDS-PAGE. The isoelectric points are reported at 25°C.1... 

---