Characterization of Fluorescently Labeled Protein with Electrospray Ionization-MS and Fluorescence Spectroscopy: How Random is Random Labeling?

Solvent exposed lysine residues are abundantly present in many proteins. Their highly reactive ε-amino groups serve as universal targets for coupling with active esters of various extrinsic labels including a vast arsenal of fluorescent probes. Here, we describe fluorescent properties and preferential localization of two frequently used fluorescent labels, AlexaFluor488 (AF488) and Cy3, on the surface of a small highly soluble serum protein neutrophil gelatinase-associated lipocalin (NGAL), which serves as a diagnostic marker for acute kidney failure. Using a standard protocol for labeling with either AF488-SDP or AF488-NHS, we achieved >95% labeling efficiency of the protein as determined by UV-vis absorption and electrospray ionization (ESI)-MS. However, fluorescence intensity of the labeled protein was less than 10% of the expected value. To understand the unusually high quenching of the probe, we identified the sites of AF488 attachments by means of LC-MS/MS combined with trypsin digestion. Surprisingly, we found that the AF488 label is not randomly distributed among accessible lysines but predominantly bound to the residues K125, K126, or K135, which are located in the NGAL calyx and are likely quenched by neighboring tryptophans and tyrosines. In contrast, when NGAL was labeled with Cy3, the probe's fluorescence was almost fully retained. The LC-MS/MS data indicated that Cy3 was predominately bound to another lysine, K31, on the protein surface on the opposite side of the calyx. Our findings suggest that a combination of the inherent properties of the label and the specifics of the protein microenvironment may selectively lead probes to specific lysine residues and thus challenge the common view that protein labeling is a random process.

Introduction of the mass spread function for characterization of protein conjugates.

Traditionally, characterization of protein molecules conjugated with molecular probes is performed by UV-vis spectroscopy. This method determines the average incorporation ratio but does not yield information about the label distribution. Electrospray ionization mass spectroscopy (ESI-MS) allows direct measurement of the fraction of protein containing a given number of labels. However, for a glycosylated protein, this analysis can be severely limited due to spectral overlap of the labels and carbohydrates. To address this problem, we introduce the mass spread function (MSF) for conjugation analysis. By treating the ESI-MS spectrum of conjugated protein as the spectrum before conjugation convolved with the MSF, we are able to quantify the labeled protein population using a binomial distribution function. We first applied this procedure for characterization of labeled antibody F(ab')(2) fragments which do not contain carbohydrates. We then apply the MSF to fit spectra of entire conjugated monoclonal antibodies and quantify the distribution of labels in the presence of glycans.

Development of an Abbott ARCHITECT cyclosporine immunoassay without metabolite cross-reactivity.

OBJECTIVE: We investigated the mechanism by which the ARCHITECT cyclosporine (CsA) chemiluminescent microparticle immunoassay (CMIA) eliminates cross-reactivity to CsA metabolites AM1 and AM9, despite its use of a monoclonal antibody which shows cross-reactivity in fluorescence polarization immunoassays. DESIGN AND METHODS: The CMIA was accomplished by incubating an extracted blood sample with magnetic microparticles coated with a very low amount of anti-CsA antibody. After a wash step the microparticles were incubated with a chemiluminescent CsA tracer, followed by a second wash step and measurement of chemiluminescence. The reagent concentrations of salt and detergent were optimized to maximize CsA binding and minimize metabolite interference. RESULTS: Elimination of CsA metabolite cross-reactivity was shown using purified metabolites and blood samples containing native CsA metabolites. The CMIA demonstrated precision and sensitivity acceptable for use in a clinical setting. CONCLUSION: We conclude that it is possible to eliminate CsA metabolite immuno-cross-reactivity by careful assay design.

State-of-the-art of serum testosterone measurement by isotope dilution-liquid chromatography-tandem mass spectrometry.

BACKGROUND: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC-MS/MS procedures in a method comparison with an ID-gas chromatography (GC)-MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine. METHODS: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC-MS/MS) and quadruplicate (ID-GC-MS) in independent runs. RESULTS: The testosterone concentrations by ID-GC-MS were 0.2-4.4 nmol/L (women), 0.2-2.0 nmol/L (hypogonadal man), and 10.1-31.3 nmol/L (normogonadal men). For ID-GC-MS, the CV was nearly constant, with a median of 1.0%; for ID-LC-MS/MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90-1.11, -0.055-0.013 nmol/L, and 0.993-0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC-MS between -9.6 and 0.4%. CONCLUSIONS: This study demonstrated fairly good accuracy and standardization of the tested ID-LC-MS/MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC-MS/MS procedures.

Performance characteristics of eight estradiol immunoassays.

Measurement of estradiol is useful in assisted reproduction, evaluation of infertility, menopause, and male feminization. The analytic performance of 8 estradiol immunoassays was evaluated. The imprecision and accuracy of the Access, ADVIA Centaur, ARCHITECT i2000, AutoDELFIA, Elecsys 2010, IMMULITE 2000, and Vitros ECi estradiol assays (see text for proprietary information) were evaluated by using an isotope dilution-gas chromatography-mass spectrometry (ID-GC-MS) reference method. The coefficient of variation (CV) ranged from 6.9% on the Elecsys 2010 to 42.6% on the ADVIA Centaur at an estradiol concentration of 18 pg/mL (66 pmol/L), with the ARCHITECT i2000 assay in development and the Vitros ECi having a CV below 10% at this estradiol concentration. Agreement between the automated assays and ID-GC-MS was variable, with slopes ranging from 0.87 to 1.20. The Access, ARCHITECT i2000 in development, and the IMMULITE 2000 were the most accurate, with slopes of 0.99, 0.98, and 1.03, respectively. These findings indicate that the ARCHITECT i2000 estradiol assay in development had the best precision and accuracy of the assays evaluated for measurement of serum estradiol concentrations.