Magnetic resin composites for the enrichment of proteins, peptides and phosphopeptides.

There is growing interest in the development of materials for enriching proteins and phosphoproteins from complex sample matrices for mass spectrometric analysis. Herein, we designed and synthesized two types of magnetic resin composites, i.e., MTS9200@Fe3O4 and FPA90CL@Fe3O4, and assessed their applications as adsorbents for enriching proteins, peptides and phosphopeptides. With the combination of Fe3+-IMAC interaction (MTS9200) or electrostatic attraction (FPA90CL) of resins and the adsorption of Fe3O4, the prepared composites exhibited higher capacities for adsorbing a protein (bovine serum albumin, at 195.71 and 135.03 mg g-1 for MTS9200@Fe3O4 and FPA90CL@Fe3O4, respectively) than MTS9200, FPA90CL and Fe3O4. In addition, due to the contributions of the hydrophobic skeleton of resins and Fe3O4, the magnetic resin composites allowed for efficient enrichment of peptides. Moreover, through Fe3+-IMAC interaction or electrostatic attraction of resins and Fe-O MOAC interaction of Fe3O4 with phosphate groups, phosphopeptides could also be captured. Furthermore, we employed the prepared composites for enriching proteins and phosphopeptides from human serum, where 466 and 506 proteins, and 434 and 356 phosphorylation sites, were detected from human serum after being processed with FPA90CL@Fe3O4 and MTS9200@Fe3O4, respectively. Together, our work revealed the great potential of magnetic resin composites as enrichment materials for proteomics and phosphoproteomics analysis.

In situ synthesis and dynamic simulation of molecularly imprinted polymeric nanoparticles on a micro-reactor system.

Current practices in synthesizing molecularly imprinted polymers face challenges-lengthy process, low-productivity, the need for expensive and sophisticated equipment, and they cannot be controlled in situ synthesis. Herein, we present a micro-reactor for in situ and continuously synthesizing trillions of molecularly imprinted polymeric nanoparticles that contain molecular fingerprints of bovine serum albumin in a short period of time (5-30 min). Initially, we performed COMSOL simulation to analyze mixing efficiency with altering flow rates, and experimentally validated the platform for synthesizing nanoparticles with sizes ranging from 52-106 nm. Molecular interactions between monomers and protein were also examined by molecular docking and dynamics simulations. Afterwards, we benchmarked the micro-reactor parameters through dispersity and concentration of molecularly imprinted polymers using principal component analysis. Sensing assets of molecularly imprinted polymers were examined on a metamaterial sensor, resulting in 81% of precision with high selectivity (4.5 times), and three cycles of consecutive use. Overall, our micro-reactor stood out for its high productivity (48-288 times improvement in assay-time and 2 times improvement in reagent volume), enabling to produce 1.4-1.5 times more MIPs at one-single step, and continuous production compared to conventional strategy.

Development of novel enzyme immobilization methods employing formaldehyde or triethoxysilylbutyraldehyde to fabricate immobilized enzyme microreactors for peptide mapping.

The digestion of proteins with proteolytic enzymes has expedited the analysis of peptide mapping. Here, we compared the digestion efficiency of soluble chymotrypsin (CT) with two immobilized CT preparations using bovine serum albumin (BSA) as the substrate. An efficient method of immobilizing chymotrypsin using formaldehyde (FA) was optimized and the conditions were applied to assess a novel immobilization reagent, triethoxysilylbutaraldehyde (TESB). Efforts to determine the best enzyme-to-substrate (E : S) ratios during digestion of denatured BSA with single-use FA-CT enzyme particles were performed by adjusting the amount of substrate used. An E : S ratio of 10 : 1 was found to be best based on the LC-MS/MS analysis data showing sequence coverage of 67%. Fabrication of immobilized enzyme microreactors (IMERs) was carried out using both (3-aminopropyl)triethoxysilane (APTES) with the idealized conditions with FA, as well as the novel procedure utilizing TESB for a proof of concept open-tubular IMER. It was found that the FA-APTES IMER had a sequence coverage of 6%, while the TESB IMER had 29% sequence coverage from MS analysis. The application of TESB in enzyme immobilization has the potential to facilitate a greater degree of enzymatic digestion with higher sequence coverage than traditional immobilization or crosslinking reagents for bottom-up proteomics.

The proteomic and particle composition of human platelet lysate for cell therapy products.

Following health agencies warning, the use of animal origin supplements should be avoided in biological products proposed as therapy in humans. Platelet lysate and several other growth factors sources are alternatives to replace fetal calf serum, the current gold standard in clinical-grade cell culture. However, the platelet supplement's content lacks data due to different production methods. The principle behind these products relays on the lysis of platelets that release several proteins, some of which are contained in heterogeneous granules and coordinate biological functions. This study aims to analyze the composition and reproducibility of a platelet lysate produced with a standardized method, by describing several batches' protein and particle content using proteomics and dynamic light scattering. Proteomics data revealed a diversified protein content, with some related to essential cellular processes such as proliferation, morphogenesis, differentiation, biosynthesis, adhesion, and metabolism. It also detected proteins responsible for activation and binding of transforming growth factor beta, hepatocyte growth factor, and insulin-like growth factor. Total protein, biochemical, and growth factors quantitative data showed consistent and reproducible values across batches. Novel data on two major particle populations is presented, with high dispersion level at 231 ± 96 d.nm and at 30 ± 8 d.nm, possibly being an important way of protein trafficking through the cellular microenvironment. This experimental and descriptive analysis aims to support the content definition and quality criteria of a cell supplement for clinical applications.

Highly efficient enrichment of intact phosphoproteins by a cadmium ion-based co-precipitation strategy.

Selective separation and enrichment of phosphoproteins are essential for understanding their important functions in almost all cellular processes. Here, taking advantage of the feature that cadmium ion (Cd2+ ) has an overwhelming preference for phosphates, we developed a robust and simple Cd2+ co-precipitation strategy for the selective isolation of intact phosphoproteins. After evaluating the feasibility of Cd2+ in phosphoprotein precipitation, we compared the washing protocols for the removal of non-specific binding proteins and then used the best-performing protocol for the isolation of phosphoproteins from different complex samples. It was found that phosphoproteins can be specifically enriched from artificial protein mixtures containing α-casein, ß-casein, and bovine serum albumin or plasma, in which bovine serum albumin or plasma were served as interferences with very high molar ratios. Applying this method to enrich phosphoproteins from complex cell lysates, a high specificity was confirmed by western blotting analysis with a phosphoprotein-specific kit. Finally, we successfully applied this method to the purification of caseins from drinking milk, highlighting its potential application in the studies where purified phosphoproteins were required. In a word, this Cd2+ co-precipitation method enables universal and effective capture, enrichment, and detection of intact phosphoproteins, making it a powerful tool for the comprehensive analysis of the phosphoproteome.

Optically active plasmonic cellulose fibers based on Au nanorods for SERS applications.

Cellulose might be a promising material for surface-enhanced Raman scattering (SERS) substrates due to its wide availability, low cost, ease of fabrication, high flexibility and low optical activity. This work shows, for the first time development of the cellulose-based substrate, that owes its SERS activity to the presence of gold nanorods in its internal structure, and not only on the surface, as it is shown elsewhere, thus ensuring superior stability of the obtained material. This flexible cellulose-based substrate exhibiting plasmonic activity, provide easy and reproducible detection of different analytes via SERS technique. The substrate was prepared by introduction of gold nanorods into the cellulose fibers matrix using an eco-friendly process based on N-Methylmorpholine-N-Oxide. Au-modified cellulose fibers were used for the detection of p-Mercaptobenzoic acid and Bovine Serum Albumin by the SERS method. The obtained results show that this substrate offers large signal enhancement of 6-orders of magnitude, and high signal reproducibility with a relative standard deviation of 8.3%. Additionally, washing tests (90 °C, 20 h) showed superior stability of the as prepared plasmonic fibers, thus proving the good reusability of the substrates and the long shelf life.

Fourier spotting: a novel setup for single-color reflectometry.

Single-color reflectrometry is a sensitive and robust detection method in optical biosensor applications, for example for bioanalysis. It is based on the interference of reflected monochromatic radiation and is label free. We present a novel setup for single-color reflectometry based on the patented technology of Berner et al. from 2016. Tilting areas of micro-mirrors allow us to encode the optical reflection signal of an analyte and reference channel into a particular carrier frequency with the amplitude being proportional to the local reflection. Therefore, a single photodiode is sufficient to collect the signals from both channels simultaneously. A 180∘ phase shift in the tilt frequency of two calibrated micro-mirror areas leads to a superposition of the analyte and reference signal which enables an efficient reduction of the baseline offset and potential baseline offset drift. A performance test reveals that we are able to detect changes of the refractive index n down to Δn < 0.01 of saline solutions as regents. A further test validates the detection of heterogeneous binding interaction. This test compromises immobilized testosterone-bovine serum albumin on a three-dimensional layer of biopolymer as ligand and monoclonal anti-testosterone antibodies as analyte. Antibody/antigen binding induces a local growth of the biolayer and change in the refractive index, which is measured via the local change of the reflection. Reproducible measurements enable for the analysis of the binding kinetics by determining the affinity constant KA = 1.59 × 10- 7 M- 1. In summary, this work shows that the concept of differential Fourier spotting as novel setup for single-color reflectometry is suitable for reliable bioanalysis. Graphical Abstract.

Raman spectroscopy as a process analytical technology to investigate biopharmaceutical freeze concentration processes.

Freezing processes are a well-established unit operation in the biopharmaceutical industry to increase the shelf-life of protein-based drugs. While freezing reduces degradation reaction rates, it may also exert stresses such as freeze concentration. Macroscopic freeze concentration in large-scale freezing processes has been described thoroughly by examination of frozen bulk material, but the transient process leading to such freeze concentration profiles has not been monitored yet for biopharmaceutical solutions. In this study, Raman spectroscopy as a process analytical technology is demonstrated for model formulations containing monoclonal antibodies (mAbs) or bovine serum albumin (BSA) in varying concentrations of sucrose and buffer salts. Therefore, a Raman probe was immersed into a bulk volume at different heights, monitoring the freeze concentration in the liquid phase during the freezing processes. Partial least square regression models were used to quantitatively discriminate between the protein and excipients simultaneously. The freeze concentration profiles were dependend on freezing temperature and formulation with freeze concentrations up to 2.4-fold. Convection currents at the bottom of the freezing container were observed with a maximum height of 1 mm. Furthermore, freeze concentration was correlated with the sucrose concentration in a formulation. Analysis of the freeze concentration slope indicated diffusion from the bottom to the top of the container. In summary, Raman spectroscopy is a valuable tool for process validation of freeze concentration simulations and to overcome scale-dependent challenges.

Squaraine dyes as serum albumins probes: Synthesis, photophysical experiments and molecular docking studies.

Three barbiturate squaraine dyes derived from indolenine or benzothiazole, with different barbituric acid derivatives were prepared, characterized and photophysically evaluated by standard spectroscopic methods. As expectable for squaraines, these dyes showed narrow and intense absorption and emission bands in the Vis/NIR region. The interaction of synthesized dyes with bovine and human serum albumins (BSA and HSA) was also evaluated in phosphate buffer (PB). The results revealed that upon the addition of BSA or HSA the complex dye-protein emit more fluorescence, and the emission intensity is directly proportional to the concentration of protein used (0-3.5 µM). The titration tests allowed to calculate the binding constants, in an order of magnitude of 104-106 M, as well as the limits of detection and quantification in the nanomolar tens range. All dyes showed a good response to the interaction with both proteins, but the most pronounced envisioning their use as protein labeling was observed for the squaraine dye derived from the indolenine with a 1,3-dimethylbarbituric acid moiety. The molecular docking studies revealed the existence of a binding between the compounds and four sites on the HSA molecule, where one of these four locations is a new binding site with which this series of dye interacts.

Removal of protein wastes by cylinder-shaped NaY zeolite adsorbents decorated with heavy metal wastes.

Cylinder-shaped NaY zeolite was used as an adsorbent for eradicating both heavy metal ions (Cu2+, Zn2+, Ni2+, and Co2+) and proteins from the waste streams. As a pseudo-metal ion affinity adsorbent, NaY zeolite was used in the capture of heavy metal ions in the first stage. The amount (molar basis) of metal ions adsorbed onto NaY zeolite decreased in the order of Cu2+ > Zn2+ > Co2+ > Ni2+. Bovine serum albumin (BSA) was utilized as a model of proteins used in the waste adsorption process by NaY zeolite. The adsorption capacities of NaY zeolite and Cu/NaY zeolite for BSA were 14.90 mg BSA/g zeolite and 84.61 mg BSA/g zeolite, respectively. Moreover, Cu/NaY zeolite was highly stable in the solutions made of 2 M NaCl, 500 mM imidazole or 125 mM EDTA solutions. These conditions indicated that the minimal probability of secondary contamination caused by metal ions and soluble proteins in the waste stream. This study demonstrates the potential of Cu/NaY zeolite complex as an efficient pseudo-metal chelate adsorbent that could remove metal ions and water-soluble proteins from wastewater concurrently.

Comparison of Single- and Mixed-Sized Gold Nanoparticles on Lateral Flow Assay for Albumin Detection.

The sensitivity and reproducibility of the lateral flow assay can be influenced by multiple factors, such as the size of gold nanoparticles (GNPs) employed. Here, we evaluated the analytical performance of single-sized and mixed-sized GNPs using a simple lateral flow assay (LFA) platform. This platform was used as a model assay to diagnose albumin levels and demonstrate the analytical performance of single-sized and mixed-sized GNPs in LFA tests. Two sizes of GNPs@anti-bovine serum albumin (BSA) conjugate proteins were mixed at different ratios. The unique optical properties of the GNPs induced a distinguishing color-shedding effect on the single- and mixed-sized GNPs@anti-BSA conjugates interacting with the target analyte BSA spotted on the test line. The use of mixed-sized GNPs@anti-BSA conjugates enhanced signal relative to the 20 nm GNPs, and provided superior stability compared with solely employing the large GNPs (50 nm). The proposed platform in this study could provide an efficient BSA detection mechanism that can be utilized as a model biomarker for confronting chronic kidney disease.

Trinuclear Organometallic Pt-Ir-Pt Complexes: Insights into Photophysical Properties, Amino Acid Binding and Protein Sensing.

The rational synthesis of trinuclear emissive organometallic complexes with two equivalent platinum(II) centres appended to the ancillary substituted 2,2'-bipyridyl ligand of the cyclometalated iridium(III) centre is reported here. The alkynyl-platinum moiety and cyclometalated iridium(III) centres have been separated through a non-conjugated CH2 -O-CH2 linkage. The emission titration with amino acids reveals that the complexes sense free amino acids. The luminescence sensing of BSA is thus attributed to the amino acid sensing ability of the complexes and confirmed by emission anisotropy and Far-UV CD spectral study. The decrease in α-helix in the CD spectra signifies the changes in the secondary structure of protein in presence of the complexes.

Metal-Organic Framework-Derived Hollow and Hierarchical Porous Multivariate Metal-Oxide Microspheres for Efficient Phosphoproteomics Analysis.

Pre-enrichment of the biological samples is a crucial step in phosphoproteomics research. At present, metal-oxide affinity chromatography (MOAC) is one of the most recognized enrichment strategy. Therefore, the design and preparation of a MOAC-based affinity material with better enrichment properties will be of great significance for the phosphoproteomics study. In this work, we obtained a novel multivariate metal-oxide microsphere (NiFe2O4@C@TiO2) with a hollow and hierarchical porous structure through pyrolysis of TiO2-modified Fe/Ni-based metal-organic frameworks (MOFs). After pyrolysis, the carbon matrix derived from the MOFs provided support and porous properties. Meanwhile, multivariate metal oxides endowed the microspheres with an excellent magnetic response property and superior enrichment performance for phosphorylated biomolecules. The unique hollow and hierarchical porous structure greatly enhanced the diffusion of phosphorylated biomolecules. Therefore, the microspheres exhibited excellent enrichment performance for phosphorylated biomolecules: a large adsorption capacity (124 µmol g-1), excellent selectivity (α-casein/BSA, 1:5000, m/m), perfect size-exclusion performance (α-casein digests/α-casein/BSA, 1:500:500), and extremely low detection limit (2 fmol). Furthermore, the microspheres showed excellent enrichment performance in a series of real biological samples, such as nonfat milk, serum, saliva, rat brain tissue, and plasma exosomes of patients with esophageal cancer, which further demonstrated its huge application potential in MS-based phosphoproteomics research.

Dissociation of Biomolecules by an Intense Low-Energy Electron Beam in a High Sensitivity Time-of-Flight Mass Spectrometer.

We report the progress on an electron-activated dissociation (EAD) device coupled to a quadrupole TOF mass spectrometer (QqTOF MS) developed in our group. This device features a new electron beam optics design allowing up to 100 times stronger electron currents in the reaction cell. The electron beam current reached the space-charge limit of 0.5 µA at near-zero electron kinetic energies. These advances enable fast and efficient dissociation of various analytes ranging from singly charged small molecules to multiply protonated proteins. Tunable electron energy provides access to different fragmentation regimes: ECD, hot ECD, and electron-impact excitation of ions from organics (EIEIO). The efficiency of the device was tested on a wide range of precursor charge states. The EAD device was installed in a QqTOF MS employing a novel trap-and-release strategy facilitating spatial mass focusing of ions at the center of the TOF accelerator. This technique increased the sensitivity 6-10 times and allows for the first time comprehensive structural lipidomics on an LC time scale. The system was evaluated for other compound classes such as intact proteins and glycopeptides. Application of hot ECD for the analysis of glycopeptides resulted in rich fragmentation with predominantly peptide backbone fragments; however, glycan fragments attributed to the ECD process were also observed. A standard small protein ubiquitin (8.6 kDa) was sequenced with 90% cleavage coverage at spectrum accumulation times of 100 ms and 98% at 800 ms. Comparable cleavage coverage for a medium-size protein (carbonic anhydrase: 29 kDa) could be achieved, albeit with longer accumulation times.

Signal Amplification in an Optical and Dielectric Biosensor Employing Liquid Crystal-Photopolymer Composite as the Sensing Medium.

An optical and dielectric biosensor based on a liquid crystal (LC)-photopolymer composite was established in this study for the detection and quantitation of bovine serum albumin (BSA). When the nematic LC E7 was doped with 4-wt.% NOA65, a photo-curable prepolymer, and photopolymerized by UV irradiation at 20 mW/cm2 for 300 s, the limit of detection determined by image analysis of the LC optical texture and dielectric spectroscopic measurements was 3400 and 88 pg/mL for BSA, respectively, which were lower than those detected with E7 alone (10 µg/mL BSA). The photopolymerized NOA65, but not the prepolymer prior to UV exposure, contributed to the enhanced optical signal, and UV irradiation of pristine E7 in the absence of NOA65 had no effect on the optical texture. The effective tilt angle θ, calculated from the real-part dielectric constant ε', decreased with increasing BSA concentration, providing strong evidence for the correlation of photopolymerized NOA65 to the intensified disruption in the vertically oriented LC molecules to enhance the optical and dielectric signals of BSA. The optical and dielectric anisotropy of LCs and the photo-curable dopant facilitate novel quantitative and signal amplification approaches to potential development of LC-based biosensors.

Production and characterization of antibody against Opisthorchis viverrini via phage display and molecular simulation.

In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti-O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti-O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti-O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues.

Proof-of-concept analytical instrument for label-free optical deconvolution of protein species in a mixture.

The adoption of process analytical technologies by the biopharmaceutical industry can reduce the cost of therapeutic drugs and facilitate investigation of new bioprocesses. Control of critical process parameters to retain critical product quality attributes within strict bounds is important for ensuring a consistently high product quality, but developing the sophisticated analytical technologies required has proven to be a major challenge. Here, we demonstrate a new optical technique for continuous monitoring of protein species as they are eluted from a chromatographic column, even when they fully co-elute with other protein species, without making any assumption about or peak-fitting to the elution profile. To achieve this, we designed and constructed a time-resolved intrinsic fluorescence lifetime chromatograph, and established an analytical framework for deconvolving and quantifying distinct but co-eluting protein species in real time. This proof-of-concept technology has potentially useful applications as a process analytical technology and more generally as an analytical technique for label-free quantification of proteins in mixtures.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Exploiting the Catalytic Ability of Polydopamine-Remodeling Gold Nanoparticles toward the Naked-Eye Detection of Cancer Cells at a Single-Cell Level.

In this study, a catalytic polydopamine-remodeling gold nanoparticle sensitized with an antinucleolin AS1411 probe (pAu nanoprobe) is synthesized, where the surface of the gold nanoparticles (AuNPs) is modified with a spontaneous self-polymerization of a polydopamine coating that imparts the probe functionalization ability and antispecific protein binding while the intrinsic catalytic property of the AuNPs is preserved. The functionalized AS1411 probe exerts specific recognition with nucleolin protein that is found to be overexpressed on the surface of breast cancer cells (MDA-MB-231). Scanning electron microscopy (SEM) confirms that the specific binding of the pAu nanoprobe occurs at the cancer cell surface. Taking advantage of the catalytic ability of the pAu nanoprobe in reducing blue-colored methylene blue (MB) to colorless leuco-MB, a colorimetric biosensing platform is established based on the accessible catalytic active sites on the pAu nanoprobe toward MB. The specific binding inhibits the pAu nanoprobe from efficiently catalyzing the reduction of MB, resulting in a "turn-off" catalytic biosensing platform. The catalytic conversion of MB is inversely proportional to the concentration of the nucleolin protein and the cancer cells, yielding a detection limit of 15 pM of the nucleolin protein and two cancer cells. The presence of five orders of magnitude higher concentration of bovine serum albumin hardly affects the catalytic ability of the pAu nanoprobe, that is, 88% catalytic ability is still preserved, which validates the specificity of the proposed pAu nanoprobe. In particular, a distinct color contrast creates a significant signal-to-noise ratio so as to enable single-cell level detection of two cancer cells by naked-eye judgment. Moreover, the undiluted, real human serum samples spiked with the cancer cells were examined with an impressive recovery of 94 ± 0.3%, which holds great promise in cancer cell screening.

Development and validation of an RP-HPLC DAD method for the simultaneous quantification of minor and major whey proteins.

Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, ß-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% - 103%) and recovery (96% - 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min.