Mobile Affinity Selection Chromatography Analysis of Therapeutic Monoclonal Antibodies.

Federal regulatory agencies require continuous verification of recombinant therapeutic monoclonal antibody (mAb) quality that is commonly achieved in a two-step process. First, the host-cell proteome and metabolome are removed from the production medium by protein A affinity chromatography. Second, following recovery from the affinity column with an acidic wash, mAb quality is assessed in multiple ways by liquid chromatography-mass spectrometry (LC-MS). However, lengthy sample preparation and the lack of higher-order structure analyses are limitations of this approach. To address these issues, this report presents an integrated approach for the analysis of two critical quality attributes of mAbs, namely titer and relative aggregate content. Integration of sample preparation and molecular-recognition-based analyses were achieved in a single step utilizing an isocratically eluted mobile affinity selection chromatography (MASC) column. MASC circumvents the protein A step, simplifying sample preparation. Within 10 min, (i) mAbs are fluorescently coded for specific detection, (ii) monomers and aggregates are resolved, (iii) the mAb titer is quantified, (iv) relative aggregate content is determined, (v) analytes are detected, and (vi) the column is ready for the next sample. It is suggested herein that this mode of rapid quality assessment will be of value at all stages of discovery (screening, clone selection, characterization), process R&D, and manufacturing. Rapid monitoring of variant formation is a critical element of quality evaluation.

Mobile Affinity Sorbent Chromatography.

The objective in routine analyses is generally to determine a small number of analytes. With samples containing ∼103 or more components there will be insufficient peak capacity to resolve analytes from nonanalytes. This issue was addressed herein through a new type of separation mechanism in which small groups of targeted analytes are bound with high affinity to a soluble analyte-sequestering transport phase (ASTP) composed of a ∼25 nm Stokes radius hydrophilic polymer core (HPC). When introduced into a 30 nm pore diameter size-exclusion chromatography (SEC) column, ASTP/analyte complexes elute within minutes, together, unretained, and relatively pure in the first chromatographic peak. Nonanalytes, in contrast, enter pore matrices of the packing material, are retarded in elution velocity, and are eluted later, separated from analytes. Fabrication of ASTPs was achieved by covalently coupling an antibody or some other affinity selector to a high molecular weight HPC. Beyond sequestering analytes, the function of ASTPs is to act as a molecular weight shifting agent, conveying an effective molecular weight to analytes that is much larger than that of nonanalytes and causing them to elute in the SEC void volume. This mode of separation is referred to as mobile affinity sorbent chromatography (MASC). Subsequent to their purification, ASTP/analyte complexes were detected by fluorescence spectrometry.

Smartphone-based colorimetric analysis for detection of saliva alcohol concentration.

A simple device and associated analytical methods are reported. We provide objective and accurate determination of saliva alcohol concentrations using smartphone-based colorimetric imaging. The device utilizes any smartphone with a miniature attachment that positions the sample and provides constant illumination for sample imaging. Analyses of histograms based on channel imaging of red-green-blue (RGB) and hue-saturation-value (HSV) color space provide unambiguous determination of blood alcohol concentration from color changes on sample pads. A smartphone-based sample analysis by colorimetry was developed and tested with blind samples that matched with the training sets. This technology can be adapted to any smartphone and used to conduct color change assays.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma.

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Accelerating trypsin digestion: the immobilized enzyme reactor.

Sample preparation has lagged far behind the evolution of instrumentation used in mass-linked protein analysis. Trypsin digestion, for example, still takes a day, as it did 50 years ago, while mass spectral analyses are achieved in seconds. Higher order structure of proteins is frequently modified by varying digestion conditions: shifting the initial points of trypsin cleavage, changing digestion pathways, accelerating peptide bond demasking and altering the distribution of miscleaved products at the completion of proteolysis. Reduction and alkylation are even circumvented in many cases. This review focuses on immobilized enzyme reactor technology as a means to achieve accelerated trypsin digestion by exploiting these phenomena.

Simple, miniaturized blood plasma extraction method.

A rapid plasma extraction technology that collects a 2.5 µL aliquot of plasma within three minutes from a finger-stick derived drop of blood was evaluated. The utility of the plasma extraction cards used was that a paper collection disc bearing plasma was produced that could be air-dried in fifteen minutes and placed in a mailing envelop for transport to an analytical laboratory. This circumvents the need for venipuncture and blood collection in specialized vials by a phlebotomist along with centrifugation and refrigerated storage. Plasma extraction was achieved by applying a blood drop to a membrane stack through which plasma was drawn by capillary action. During the course of plasma migration to a collection disc at the bottom of the membrane stack blood cells were removed by a combination of adsorption and filtration. After the collection disc filled with an aliquot of plasma the upper membranes were stripped from the collection card and the collection disc was air-dried. Intercard differences in the volume of plasma collected varied approximately 1% while volume variations of less than 2% were seen with hematocrit levels ranging from 20% to 71%. Dried samples bearing metabolites and proteins were then extracted from the disc and analyzed. 25-Hydroxy vitamin D was quantified by LC-MS/MS analysis following derivatization with a secosteroid signal enhancing tag that imparted a permanent positive charge to the vitamin and reduced the limit of quantification (LOQ) to 1 pg of collected vitamin on the disc; comparable to values observed with liquid-liquid extraction (LLE) of a venipuncture sample. A similar study using conventional proteomics methods and spectral counting for quantification was conducted with yeast enolase added to serum as an internal standard. The LOQ with extracted serum samples for enolase was 1 µM, linear from 1 to 40 µM, the highest concentration examined. In all respects protein quantification with extracted serum samples was comparable to that observed with serum samples obtained by venipuncture.

Integrated sample preparation methodology for proteomics: analysis of native proteins.

An innovative sample preparation strategy is reported for protein identification in complex mixtures based on integration of affinity chromatographic selection and accelerated trypsin digestion using a continuous flow immobilized enzyme reactor (cf-IMER). Affinity selected glycoproteins were released to a cf-IMER column which converted native proteins to peptides in 5 min at elevated temperature. Digestion with the cf-IMER was compared to the traditional 16 h solution-based trypsin digestion of reduced and alkylated proteins. With immobilized antibody selection of Lewis x (Le(x)) glycan bearing glycoproteins from plasma, 66 proteins were identified in total with the two methods while approximately 1/3 of the total proteins and peptides were only observed with the cf-IMER. This suggests that proteomics based on protein identification by reduction and alkylation with solution-based trypsin digestion alone may not be identifying large numbers of proteins or peptides present at detectable levels in samples. Furthermore, except for proteins containing a high content of disulfide bonds, the majority of proteins did not require reduction and alkylation steps for their identification. The validity of the proposed proteolysis was evaluated in several ways by analyses of a model protein and yeast lysates where the reproducibility of quantification was essentially the same with both cf-IMER and solution-based proteolysis.

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin.

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin-IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin- IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.

Native protein proteolysis in an immobilized enzyme reactor as a function of temperature.

Trypsin concentration and the unmasking of cleavage sites in proteins play important roles in the stoichiometry of peptide production and the number of limit peptides generated during proteolysis. The hypothesis explored in this work was that native proteins could be digested and identified without disulfide reduction by (i) enhancing the unmasking of cleavage sites through elevated reaction temperatures and (ii) increasing trypsin concentration by use of an immobilized enzyme reactor (IMER). Transferrin was chosen as a model protein for these studies on the basis of its resistance to trypsin digestion. Results from this study showed greater than 70% sequence coverage in the peptides identified when nonreduced transferrin was digested at 60 °C. Large numbers of missed cleavages were observed from specific regions in proteins. Proteolysis appeared to start at a small number of high frequency cleavage sites in the cases of both reduced and nonreduced transferrin. Although approximately the same number of peptides were obtained from both structural forms of transferrin, the location of high frequency cleavage sites and the peptides produced were very different. Results from this study suggest that the location of initial cleavage sites along with the path of subsequent digestion depends strongly on the type of treatment used to open protein structures up for proteolysis.

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers.

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.

Effect of single amino acid substitution on oxidative modifications of the Parkinson's disease-related protein, DJ-1.

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.

Determining the effects of antioxidants on oxidative stress induced carbonylation of proteins.

There is potential that the pathological effects of oxidative stress (OS) associated diseases such as diabetes could be ameliorated with antioxidants, but this will require a clearer understanding of the pathway(s) by which proteins are damaged by OS. This study reports the development and use of methods that assess the efficacy of dietary antioxidant supplementation at a mechanistic level. Data reported here evaluate the impact of green tea supplementation on oxidative stress induced post-translational modifications (OSi-PTMs) in plasma proteins of Zucker diabetic fatty (ZDF) rats. The mechanism of antioxidant protection was examined through both the type and amount of OSi-PTMs using mass spectrometry based identification and quantification. Carbonylated proteins in freshly drawn blood samples were derivatized with biotin hydrazide. Proteins thus biotinylated were selected from plasma samples of green tea fed diabetic rats and control animals by avidin affinity chromatography, further fractionated by reversed phase chromatography (RPC); fractions from the RPC column were tryptic digested, and the tryptic digest was fractionated by RPC before being identified by tandem mass spectrometry (MS/MS). Relative quantification of peptides bearing carbonylation sites was achieved for the first time by RPC-MS/MS using selective reaction monitoring (SRM). Seventeen carbonylated peptides were detected and quantified in both control and treated plasma. The relative concentration of eight was dramatically different between control and green tea treated animals. Seven of the OSi-PTM bearing peptides had dropped dramatically in concentration with treatment while one increased, indicating differential regulation of carbonylation by antioxidants. Green tea antioxidants were found to reduce carbonylation of proteins by lipid peroxidation end products most, followed by advanced glycation end products to a slightly lower extent. Direct oxidation of proteins by reactive oxygen species (ROS) was protected the least by green tea.

Differential carbonylation of proteins as a function of in vivo oxidative stress.

This study reports for the first time qualitative and quantitative differences in carbonylated proteins shed into blood as a function of increasing levels of OS. Carbonylated proteins in freshly drawn blood from pairs of diabetic and lean rats were derivatized with biotin hydrazide, dialyzed, and enriched with avidin affinity chromatography. Proteins thus selected were used in several ways. Differences between control and diabetic subjects in relative concentration of proteins was achieved by differential labeling of tryptic digests with iTRAQ reagents followed by reversed phase chromatography (RPC) and tandem mass spectrometry (MS/MS). Identification and characterization of OS induced post-translational modification sites in contrast was achieved by fractionation of affinity selected proteins before proteolysis and RPC-MS/MS. Relative quantification of peptides bearing oxidative modifications was achieved for the first time by selective reaction monitoring (SRM). Approximately 1.7% of the proteins in Zucker diabetic rat plasma were selected by the avidin affinity column as compared to 0.98% in lean animal plasma. Among the 35 proteins identified and quantified, Apo AII, clusterin, hemopexin precursor, and potassium voltage-gated channel subfamily H member 7 showed the most dramatic changes in concentration. Seventeen carbonylation sites were identified and quantified, 11 of which changed more than 2-fold in oxidation state. Three types of carbonylation were identified at these sites: direct oxidative cleavage from reactive oxygen species, glycation and addition of advanced glycation end products, and addition of lipid peroxidation products. Direct oxidation was the dominant form of carbonylation observed while hemoglobin and murinoglobulin 1 homologue were the most heavily oxidized proteins.

Oxidative stress induced carbonylation in human plasma.

The focus of this study was on the assessment of technology that might be of clinical utility in identification, quantification, characterization of carbonylation in human plasma proteins. Carbonylation is widely associated with oxidative stress diseases. Breast cancer patient samples were chosen as a stress positive case based on the fact that oxidative stress has been reported to be elevated in this disease. Measurements of 8-isoprostane in plasma confirmed that breast cancer patients in this study were indeed experiencing significant oxidative stress. Carbonyl groups in proteins from freshly drawn blood were derivatized with biotin hydrazide after which the samples were dialyzed and the biotinylated proteins subsequently selected, digested and labeled with iTRAQ™ heavy isotope coding reagent(s). Four hundred sixty proteins were identified and quantified, 95 of which changed 1.5 fold or more in concentration. Beyond confirming the utility of the analytical method, association of protein carbonylation was examined as well. Nearly one fourth of the selected proteins were of cytoplasmic, nuclear, or membrane origin. Analysis of the data by unbiased knowledge assembly methods indicated the most likely disease associated with the proteins was breast neoplasm. Pathway analysis showed the proteins which changed in carbonylation were strongly associated with Brca1, the breast cancer type-1 susceptibility protein. Pathway analysis indicated the major molecular functions of these proteins are defense, immunity and nucleic acid binding.

Glutathione-S-transferase P1 is a critical regulator of Cdk5 kinase activity.

Cyclin dependent kinase-5 (Cdk5) activity is deregulated in Alzheimer's disease (AD) and contributes to all three hallmarks: neurotoxic ß-amyloid formation, neurofibrillary tangles, and neuronal death. However, the mechanism leading to Cdk5 deregulation remains controversial. Cdk5 deregulation in AD is usually linked to the formation of p25, a proteolysis product of Cdk5 activator p35, which leads to Cdk5 mislocalization and hyperactivation. A few studies have indeed shown increased p25 levels in AD brains; however, others have refuted this observation. These contradictory findings suggest that additional factors contribute to Cdk5 deregulation. This study identified glutathione-S-transferase pi 1 (GSTP1) as a novel Cdk5 regulatory protein. We demonstrate that it is a critical determinant of Cdk5 activity in human AD brains and various cancer and neuronal cells. Increased GSTP1 levels were consistently associated with reduced Cdk5 activity. GSTP1 directly inhibits Cdk5 by dislodging p25/p35, and indirectly by eliminating oxidative stress. Cdk5 promotes and is activated by oxidative stress, thereby engaging a feedback loop which ultimately leads to cell death. Not surprisingly, GSTP1 transduction conferred a high degree of neuroprotection under neurotoxic conditions. Given the critical role of oxidative stress in AD pathogenesis, an increase in GSTP1 level may be an alternative way to modulate Cdk5 signaling, eliminate oxidative stress, and prevent neurodegeneration.

A large, consistent plasma proteomics data set from prospectively collected breast cancer patient and healthy volunteer samples.

BACKGROUND: Variability of plasma sample collection and of proteomics technology platforms has been detrimental to generation of large proteomic profile datasets from human biospecimens. METHODS: We carried out a clinical trial-like protocol to standardize collection of plasma from 204 healthy and 216 breast cancer patient volunteers. The breast cancer patients provided follow up samples at 3 month intervals. We generated proteomics profiles from these samples with a stable and reproducible platform for differential proteomics that employs a highly consistent nanofabricated ChipCube™ chromatography system for peptide detection and quantification with fast, single dimension mass spectrometry (LC-MS). Protein identification is achieved with subsequent LC-MS/MS analysis employing the same ChipCube™ chromatography system. RESULTS: With this consistent platform, over 800 LC-MS plasma proteomic profiles from prospectively collected samples of 420 individuals were obtained. Using a web-based data analysis pipeline for LC-MS profiling data, analyses of all peptide peaks from these plasma LC-MS profiles reveals an average coefficient of variability of less than 15%. Protein identification of peptide peaks of interest has been achieved with subsequent LC-MS/MS analyses and by referring to a spectral library created from about 150 discrete LC-MS/MS runs. Verification of peptide quantity and identity is demonstrated with several Multiple Reaction Monitoring analyses. These plasma proteomic profiles are publicly available through ProteomeCommons. CONCLUSION: From a large prospective cohort of healthy and breast cancer patient volunteers and using a nano-fabricated chromatography system, a consistent LC-MS proteomics dataset has been generated that includes more than 800 discrete human plasma profiles. This large proteomics dataset provides an important resource in support of breast cancer biomarker discovery and validation efforts.

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma.

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.