Quantitative assessment confirms deep proteome analysis by integrative top-down proteomics.

The goal of integrative top-down proteomics (i.e., two-dimensional gel electrophoresis [2DE] coupled with liquid chromatography and tandem mass spectrometry [LC/MS/MS]) is a routine analytical approach that fully addresses the breadth and depth of proteomes. To accomplish this, there should be no addition, removal, or modification to any constituent proteoforms. To address two-decade old claims of protein losses during front-end proteome resolution using 2DE, here we tested an alternate rehydration method for immobilized pH gradient strips prior to isoelectric focusing (IEF; i.e., faceup compared to facedown) and quantitatively assessed losses during the front-end of 2DE (rehydration and IEF). Using a well-established high-resolution, quantitative 2DE protocol, there were no detectable proteoform losses using the alternate faceup rehydration method. Although there is a <0.25% total loss of proteoforms during standard facedown rehydration, it is insignificant in terms of having any effect on overall proteome resolution (i.e., total spot count and total spot signal). This report is another milestone in integrative top-down proteomics, disproving long-held dogma in the field and confirming that quantitative front-end 2DE/LC/MS/MS is currently the only method to broadly and deeply analyze proteomes by resolving their constituent proteoforms.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Serum albumin saturation test based on non-esterified fatty acids imbalance for clinical employment.

Fatty acids are fundamental as energy and structural source to the human cells. They are not usually found free in human circulation. Alteration in fatty acids metabolism is linked to diseases such as diabetes, preeclampsia, heart disease, and some infectious diseases. Increased levels of non-esterified fatty acids (NEFA) may cause cell dysfunction and lipotoxicity. Since physiologically fatty acids are transported bound to albumin, we propose here a simple and cheap test that consists of albumin isoelectric focusing determination to measure the potential systemic NEFA cytotoxicity. For validation of this method, albumin isoelectric focusing in 51 serum samples from 40 critically ill patients and 11 controls was compared with NEFA/albumin ratios measured by HPLC. We called this approach an albumin saturation test. This test may indicate to physicians the potential NEFA lipotoxicity guiding them throughout better patient management. The albumin saturation test can point out serum albumin-NEFA saturation through a cheap assay that could be performed by any care facility.

Two-Dimensional Isoelectric Focusing OFFGEL, Micro-Fluidic Lab-on-Chip Electrophoresis and FTIR for Assessment of Long-Term Stability of rhG-CSF Formulation.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been increasingly recognized from among one of the most abundant families of biosimilars. Upon long-term storage, the rhG-CSF is subject to subtle chemical modifications that rapidly occur and, in particular, produce deaminated variants with divergent charge. Indeed, changes in charge from glutamine deamination may alter the way rhG-SCF will refold and the structure of resulting molecule. To assess this charge heterogeneity, 2-D gel electrophoresis has limited application. Recent micro-fluidic- based technical advances offer a great alternative method to better control liquid volumes on a minute scale. Here, we used IEF OFFGEL-lab-on-chip electrophoresis for 2-D separation of the rhG-CSF peptides according to their isoelectric point (pI) and molecular weight (kDa). We used an rhG-CSF commercial therapeutic formulation, kept refrigerated 24 months after expiry. The samples were analyzed for particulate matter and charge variants. Subsequently, the secondary structure was assessed by FTIR spectroscopy and residual biological activity was recorded. Interestingly, we showed an additional band in the acidic gel area above and below the most intense protein band (fractions 10, 11, and 12 at 22.84s). This observation reveals the presence of the rhG-CSF variant charges without any additional high molecular weight impurity or biological activity decrease. We conclude that after two years of storage, the rhG-CSF solution maintained its native secondary structure with little -sheet deviation, as reflected in the 1622 cm-1 and 1695 cm-1. These data demonstrated that a combined strategy is a more suitable and accurate analytical assessment of the rhG-CSF and recombinant protein-based biosimilars.

Optimizing Analytical Depth and Cost Efficiency of IEF-LC/MS Proteomics.

IEF LC-MS/MS is an analytical method that incorporates a two-step sample separation prior to MS identification of proteins. When analyzing complex samples this preparatory separation allows for higher analytical depth and improved quantification accuracy of proteins. However, cost and analysis time are greatly increased as each analyzed IEF fraction is separately profiled using LC-MS/MS. We propose an approach that selects a subset of IEF fractions for LC-MS/MS analysis that is highly informative in the context of a group of proteins of interest. Specifically, our method allows a significant reduction in cost and instrument time as compared to the standard protocol of running all fractions, with little compromise to coverage. We develop algorithmics to optimize the selection of the IEF fractions on which to run LC-MS/MS. We translate the fraction optimization task to Minimum Set Cover, a well-studied NP-hard problem. We develop heuristic solutions and compare them in terms of effectiveness and running times. We provide examples to demonstrate advantages and limitations of each algorithmic approach. Finally, we test our methodology by applying it to experimental data obtained from IEF LC-MS/MS analysis of yeast and human samples. We demonstrate the benefit of this approach for analyzing complex samples with a focus on different protein sets of interest.

Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting.

OBJECTIVES: We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. METHODS: We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. RESULTS: Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. CONCLUSIONS: Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.

A review on preparative and semi-preparative offgel electrophoresis for multidimensional protein/peptide assessment.

Mass spectrometry (MS) techniques are commonly used for protein identification and further analysis of selected protein spots after high resolution 2-D electrophoresis. Complementary gel-free approaches have been developed during the last few years and have shown to be useful tools in modern proteomics. The development and application of various gel-free electrophoresis devices for performing protein fractionation according to the pI differences is therefore a topic of interest. This review describes the current state of isoelectric focusing (IEF) gel-free electrophoresis based on the Agilent offgel 3100 fractionator. The review includes, therefore, (i) an overview on IEF as well as other previous IEF gel-free electrophoresis developments; (ii) offgel fundamentals and future trends; (iii) advantages and disadvantages of current offgel procedures; (iv) requirements of isolated protein pellets for further offgel fractionation; (v) offgel fraction requirements to perform the second dimensional analysis by advance electrophoresis and chromatographic techniques; and (vi) effect of the offgel operating conditions on the stability of metal-protein complexes.

Quantitative pH assessment of small-volume samples using a universal pH indicator.

We developed a hue-based pH determination method to analyze digital images of samples in a 384-well plate after the addition of a universal pH indicator. The standard error of calibration for 69 pH standards was 0.078 pH units, and no sample gave an error greater than 0.23 units. We then used in-solution isoelectric focusing to determine the isoelectric point of Wnt3A protein in conditioned medium and after purification and applied the described method to assess the pH of these small-volume samples. End users may access our standard to assay the pH of their own samples with no additional calibration.

Development of chromatofocusing techniques employing mixed-mode column packings for protein separations.

Recent studies reported in the literature using mixed-mode chromatography (MMC) column packings have shown that multiple modes of interactions between the column packing and proteins can be usefully exploited to yield excellent resolution as well as salt-tolerant adsorption of the target protein. In this study, a mixed-mode separation method using commercially available column packings was explored which combines the techniques of hydrophobic-interaction chromatography and chromatofocusing. Two different column packings, one based on mercapto-ethyl-pyridine (MEP) and the other based on hexylamine (HEA) were investigated with regard to their ability to separate proteins when using internally generated, retained pH gradients. The effects of added salt and urea on the behavior of the retained pH gradient and the protein separation achieved when using MMC column packings for chromatofocusing were also investigated. Numerical simulations using methods developed in previous work were shown to agree with experimental results when using reasonable physical parameters. These numerical simulations were also shown to be a useful qualitative method to select the compositions of the starting and elution buffers in order to achieve desired shapes for the pH and ionic strength gradients. The use of the method to fractionate blood serum was explored as a prototype example application.

IQcat: multiplexed protein quantification by isoelectric QconCAT.

Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by pI to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (∼2 weeks), inexpensive and scalable purification of arg/lys labeled IQcat standards in the Escherichia coli auxotroph AT713. With this pipeline, we assess the fidelity of IQcat-based absolute quantification for ten yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.

Assessment of the quality and structural integrity of a complex glycoprotein mixture following extraction from the formulated biopharmaceutical drug product.

Biological drugs represent an important and rapidly growing class of therapeutics useful in the treatment of a variety of disorders ranging from cancer to inflammation to infectious diseases. Unlike single chemical entities, the recombinant production of these drugs in living cells confers considerable structural and chemical heterogeneity to the biologically derived protein product that constitutes the active pharmaceutical ingredient (API). In mammalian based expression systems, much of this diversity is conferred through heterogeneous protein glycosylation. These post-translational modifications can have significant effects on the structure, biological function, and pharmacological properties of the API. In addition, the bulk proteins that comprise the API are further formulated through the use of multiple excipients designed to ensure product stability, solubility, and lot-to-lot consistency. Unfortunately, these matrices can interfere with commonly available analytical methods used in the thorough chemical characterization of the biological drug product. At the same time, a demonstration of the suitable extraction of the bulk drug substance in a manner and form that does not destabilize the active ingredient or introduce any structural bias with direct reference to the original drug product is both critical and necessary. Here, we use recombinant human follicle stimulating hormone (follitropin alpha for injection) from a pharmaceutical source as an example to illustrate a suitable purification strategy to effectively extract the bulk drug substance from the formulated drug product with high purity and yield. We assess the suitability of this extraction method in preserving the structural integrity and overall quality of the drug substance relative to the formulated drug product, placing a particular emphasis on glycosylation as a key product attribute. In so doing, we demonstrate that it is possible to effectively extract the active pharmaceutical ingredient from a formulated biological drug product in a manner that is consequently sufficient for its use in comparability studies.

Screening for congenital disorders of glycosylation (CDG): transferrin HPLC versus isoelectric focusing (IEF).

OBJECTIVES: Transferrin isoelectrofocusing (Tf-IEF) is widely used to screen for Congenital Disorders of Glycosylation (CDG), but it is laborious, time-consuming, and not suitable for automation or accurate quantification. We present our experience and advantages of the implementation of Tf-HPLC. METHODS: Sera were iron saturated, lipid precipitated and filtrated on Microcon-YM10. Glycoforms were separated by HPLC on a SOURCE 15Q anion-exchange column. Detection was at 470 nm. RESULTS: We established reference values and validated the HPLC method by analysing samples with abnormal Tf-IEF. Comparison between both methods is described. CONCLUSIONS: HPLC is useful for CDG screening, especially for laboratories that deal with great number of samples, due to its easy sample processing, the possibility of performing long series of analysis and the advantage of peak quantification, which allows objective interpretations.

Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: a gel-free multidimensional electrophoresis approach for proteomic profiling--exemplified on human follicular fluid.

Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.

Serial isoelectric focusing as an effective and economic way to obtain maximal resolution and high-throughput in 2D-based comparative proteomics of scarce samples: proof-of-principle.

In 2D-based comparative proteomics of scarce samples, such as limited patient material, established methods for prefractionation and subsequent use of different narrow range IPG strips to increase overall resolution are difficult to apply. Also, a high number of samples, a prerequisite for drawing meaningful conclusions when pathological and control samples are considered, will increase the associated amount of work almost exponentially. Here, we introduce a novel, effective, and economic method designed to obtain maximum 2D resolution while maintaining the high throughput necessary to perform large-scale comparative proteomics studies. The method is based on connecting different IPG strips serially head-to-tail so that a complete line of different IPG strips with sequential pH regions can be focused in the same experiment. We show that when 3 IPG strips (covering together the pH range of 3-11) are connected head-to-tail an optimal resolution is achieved along the whole pH range. Sample consumption, time required, and associated costs are reduced by almost 70%, and the workload is reduced significantly.

Electrophoresis: the march of pennies, the march of dimes.

The present review encompasses ca. 65 years of history of developments in electrokinetic separations, taking as a starting point the year 1937, i.e. the official launching of Tiselius' moving boundary electrophoresis (MBE). The 1950s have been particularly rich in introducing novel methodologies in zone electrophoresis (ZE), thus bringing about the decline of MBE. Among them of extraordinary importance was the development of electrophoresis on agar gels coupled to immuno-diffusion at right angles, which brought a big revolution not only in biochemistry but also in clinical chemistry. Also the by now forgotten paper electrophoresis was a landmark in separation science, in that it implemented, in its "fingerprinting" version, the first genuine two-dimensional (2D) map, coupling orthogonally a charge to a hydrophobic scale separation, while permitting for the first time the detection of spot mutations, i.e. single amino acid replacements in a polypeptide chain, that paved the way to modern genetic analysis. Equally important was the introduction of starch-block electrophoresis, that brought about the notion of sieving and the first discontinuous buffers, refined, in the 1960s, by Ornstein and Davies with their classical papers combining multiphasic buffer systems to polyacrylamide gels, that went down to history as disc-electrophoresis. The 1960s also contributed with two fundamental techniques, isoelectric focusing (IEF) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) that permitted to discriminate proteins solely on the basis of surface charge and molecular mass, respectively. The 1970s gave other fundamental contributions, such as isotachophoresis, the first example of a fully instrumental approach to electrophoresis, both in its analytical and preparative version (Tachophor and Tachofrac), 2D maps combining IEF to SDS-PAGE at right angles and silver staining techniques, that incremented sensitivity by 3 orders of magnitude. The 1980s generated immobilized pH gradients and capillary zone electrophoresis (CZE), two big players that dominated the electrokinetic horizon for all the 1990s and still in vigorous use in present days. The review terminates with a glimpse, in the third millennium, onto microchip technology and hyphenated techniques, notably direct interfacing of various electrophoretic separation methods with mass spectrometry (MS).

Novel approach for large-scale, biocompatible, and low-cost fractionation of peptides in proteolytic digest of food protein based on the amphoteric nature of peptides.

A large-scale, biocompatible, and low-cost procedure for peptide fractionation based on the amphoteric nature of peptide is developed. A sample cell (120 x 100 x 50 mm) with four joint tubes (17 mm i.d. and 20 mm in length) on the front and back was prepared. On the end of the joint tubes, a nylon screen (100 mesh)-supported agarose gel layer was formed. Five or nine of the sample cells were connected. A tryptic digest of casein (2.0-3.6 L) was applied to the sample cells. At each end of the sample cell apparatus, an additional cell filled with 0.1 M H(3)PO(4) or NaOH was connected and used as anode and cathode compartments, respectively. Reproducible fractionation of peptide could be achieved by collecting fractions with specific pH values when voltage reached a plateau by applying direct current at constant power. Running time necessary for fractionation of peptide was inversely proportional to electric power and directly proportional to sample volume.

Capillary isoelectric focusing of hemoglobin variants in the pediatric clinical laboratory.

We have used capillary isoelectric focusing (cIEF) to diagnose and monitor hemoglobinopathies in children for over three years. This report describes a major revision of our original method (Clin. Chem. 1994, 40, 2288-2295) that improves the analysis of hemoglobin (Hb) variants by cIEF, especially capillary performance and quantitation precision for minor variants. The revised method uses mixed ampholytes (2% pH 6-8:3-10; 10:1), lower viscosity methylcellulose (0.375%) solution, between-sample capillary conditioning with methanol, and hemolysates prepared from red blood cells (RBC) instead of whole blood. Collectively, these changes prolonged capillary life by minimizing capillary exposure to NaOH, standardized the sample matrix, and improved the precision of peak autointegration. The between-run quantitation imprecision (% relative standard deviation) of the revised method was 0.1-3.5% for all diagnostically important major and minor Hb variants present at normal or abnormal levels. The results show the use of the revised method for (i) posttranslationally modified Hb present at low concentrations in normal blood, (ii) Hb oxidation products produced by improper sample storage, (iii) differential diagnosis of S/beta + thalassemia, G-Philadelphia trait, S/C-Harlem disease, and Hb H disease, (iv) sensitive detection of minor variants like Hb A2' as indicators of an alpha globin mutation, and (v) neonatal screening using dried blood collected on filter paper. The results show that high-efficiency separation and precise quantitation of Hb variants over a wide range of concentrations makes cIEF a comprehensive assay that can be used without adjunct analyses for the automated primary evaluation of hemoglobinopathies and thalassemias.

From image processing to classification: IV. Classification of electrophoretic patterns by neural networks and statistical methods enable quality assessment of wheat varieties for breadmaking.

The end-use quality of products made from doughs consisting of wheat flour and water is often dependent upon the storage (gluten) proteins of the grain endosperm. Today the electrophoretic patterns of the high molecular weight (HMW) glutenin subunits are used for quality selections in wheat breeding programs in several countries. In this study, we used two multivariate techniques to classify digitized patterns from isoelectric focusing of gliadins and glutenins: a two-layered neural network architecture consisting of a self-organizing feature map and a feed-forward classifier [1], and discriminant analysis [2,3]. Three groups of seven wheat varieties (Triticum aestivum L.), associated with poor, medium or good properties in relation to bread-making quality, were used. The best classification results were obtained by the neural network model, based on data from the gliadin fraction: it was possible to classify varieties associated with poor or good quality, with recognition rates of 70 and 69%, respectively. The statistical method was better suited to solve the classification problem when the data was based on the glutenin fraction: if a specific variety was already known to be non-poor, this method enabled us to classify the medium- and good-quality classes with recognition rates of 90 and 88%, respectively. The results obtained were confirmed by correlation coefficients.

Simultaneous subtyping of group specific component and esterase D by isoelectric focusing on agarose gels.

A quick, sensitive and economical technique has been developed to subtype GC and ESD simultaneously on the same agarose IEF gel. This method could be a useful tool for forensic application.