Empiricism in Microsampling: Utilizing a Novel Lateral Flow Device and Intrinsic Normalization to Provide Accurate and Precise Clinical Analysis from a Finger Stick.

BACKGROUND: Phlebotomy plays a key role in clinical laboratory medicine but poses certain challenges for the patient and the laboratory. Dried blood spots simplify collection and stabilize specimens effectively, but clinical reference intervals are based primarily on serum or plasma. We evaluated use of dried separated blood plasma specimens to simplify plasma sample collection via finger stick; however, this sampling technique posed substantial analytical challenges. We discuss herein our efforts to overcome these challenges and provide accurate and precise clinical measurements. METHODS: Microsamples of whole blood were collected via finger stick using a collection device employing laminar-flow separation of cellular blood and plasma fractions with subsequent desiccation. Samples were analyzed on modern autoanalyzers with FDA-approved reagent and calibration systems, as well as commercially available reagents with laboratory-developed assay parameters. Measured analyte concentrations from extracted dried plasma samples were normalized to a coextracted endogenous analyte, chloride. RESULTS: Chloride normalization reduced variability incurred through extraction and undefined plasma volume. Excellent correlation of normalized measurements from dried finger-stick samples (whole blood and plasma) versus matched venous samples facilitated developing mathematical transformations to provide concordance between specimen types. Independent end-to-end performance verification yielded mean biases <3% for the 5 analytes evaluated relative to venous drawn samples analyzed on FDA-approved measurement systems. CONCLUSION: Challenges inherent with this microsampling technique and alternate sample matrix were obviated through capabilities of modern autoanalyzers and implementation of chloride normalization. These results demonstrate that self-collected microsamples from a finger stick can give results concordant with those of venous samples.

Protein Biomarker Quantification by Immunoaffinity Liquid Chromatography-Tandem Mass Spectrometry: Current State and Future Vision.

Immunoaffinity-mass spectrometry (IA-MS) is an emerging analytical genre with several advantages for profiling and determination of protein biomarkers. Because IA-MS combines affinity capture, analogous to ligand binding assays (LBAs), with mass spectrometry (MS) detection, this platform is often described using the term hybrid methods. The purpose of this report is to provide an overview of the principles of IA-MS and to demonstrate, through application, the unique power and potential of this technology. By combining target immunoaffinity enrichment with the use of stable isotope-labeled internal standards and MS detection, IA-MS achieves high sensitivity while providing unparalleled specificity for the quantification of protein biomarkers in fluids and tissues. In recent years, significant uptake of IA-MS has occurred in the pharmaceutical industry, particularly in the early stages of clinical development, enabling biomarker measurement previously considered unattainable. By comparison, IA-MS adoption by CLIA laboratories has occurred more slowly. Current barriers to IA-MS use and opportunities for expanded adoption are discussed. The path forward involves identifying applications for which IA-MS is the best option compared with LBA or MS technologies alone. IA-MS will continue to benefit from advances in reagent generation, more sensitive and higher throughput MS technologies, and continued growth in use by the broader analytical community. Collectively, the pursuit of these opportunities will secure expanded long-term use of IA-MS for clinical applications.

Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised.

Stable isotopically labeled (SIL) tryptic peptides, cleavable SIL peptides, and a full-length SIL protein were compared for internal calibration (i.e., as internal calibrators) and external calibration (i.e., as internal standards) when quantifying three forms of unlabeled, human thyroglobulin (Tg) by bottom-up protein analysis. All SIL materials and human proteins were standardized by amino acid analysis to ensure traceability of measurements and allow confident assignment of accuracy. The three forms of human Tg quantified were (1) the primary reference material BCR457-a native protein purified from human thyroids, (2) a commercially available form also purified from human thyroids, and (3) a full-length recombinant form expressed and purified from a human embryonic kidney 293 cell-line. Collectively, the results unequivocally demonstrate the lack of commutability of tryptic and cleavable SIL peptides as internal calibrators across various bottom-up assays (i.e., denaturing/digestion conditions). Further, the results demonstrate the potential during external calibration for surrogate protein calibrators (i.e., recombinant proteins) to produce inaccurate concentration assignments of native protein analytes by bottom-up analysis due to variance in digestion efficiency, which is not alleviated by altering denaturation/digestion stringency and indicates why protein calibrators may not be commutable in bottom-up protein assays. These results have implications regarding the veracity of "absolute" protein concentration assignments by bottom-up assays using peptide calibrators, as well as protein calibrators, given that absolute accuracy was not universally observed. Nevertheless, these results support the use of recombinant SIL proteins as internal standards over SIL peptides due to their ability to better mimic the digestion of human-derived proteins and mitigate bias due to digestion-based matrix effects that were observed during external calibration.

Complete proteomic-based enzyme reaction and inhibition kinetics reveal how monolignol biosynthetic enzyme families affect metabolic flux and lignin in Populus trichocarpa.

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants.

Systems biology of lignin biosynthesis in Populus trichocarpa: heteromeric 4-coumaric acid:coenzyme A ligase protein complex formation, regulation, and numerical modeling.

As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein-protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.

Monolignol pathway 4-coumaric acid:coenzyme A ligases in Populus trichocarpa: novel specificity, metabolic regulation, and simulation of coenzyme A ligation fluxes.

4-Coumaric acid:coenzyme A ligase (4CL) is involved in monolignol biosynthesis for lignification in plant cell walls. It ligates coenzyme A (CoA) with hydroxycinnamic acids, such as 4-coumaric and caffeic acids, into hydroxycinnamoyl-CoA thioesters. The ligation ensures the activated state of the acid for reduction into monolignols. In Populus spp., it has long been thought that one monolignol-specific 4CL is involved. Here, we present evidence of two monolignol 4CLs, Ptr4CL3 and Ptr4CL5, in Populus trichocarpa. Ptr4CL3 is the ortholog of the monolignol 4CL reported for many other species. Ptr4CL5 is novel. The two Ptr4CLs exhibited distinct Michaelis-Menten kinetic properties. Inhibition kinetics demonstrated that hydroxycinnamic acid substrates are also inhibitors of 4CL and suggested that Ptr4CL5 is an allosteric enzyme. Experimentally validated flux simulation, incorporating reaction/inhibition kinetics, suggested two CoA ligation paths in vivo: one through 4-coumaric acid and the other through caffeic acid. We previously showed that a membrane protein complex mediated the 3-hydroxylation of 4-coumaric acid to caffeic acid. The demonstration here of two ligation paths requiring these acids supports this 3-hydroxylation function. Ptr4CL3 regulates both CoA ligation paths with similar efficiencies, whereas Ptr4CL5 regulates primarily the caffeic acid path. Both paths can be inhibited by caffeic acid. The Ptr4CL5-catalyzed caffeic acid metabolism, therefore, may also act to mitigate the inhibition by caffeic acid to maintain a proper ligation flux. A high level of caffeic acid was detected in stem-differentiating xylem of P. trichocarpa. Our results suggest that Ptr4CL5 and caffeic acid coordinately modulate the CoA ligation flux for monolignol biosynthesis.

Peptide production and decay rates affect the quantitative accuracy of protein cleavage isotope dilution mass spectrometry (PC-IDMS).

No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in protein cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent and postdigestion introduction of the SIL standards was related to differential decay experienced by the SIL peptide and the native peptide in each method. Given these results, we propose concurrent introduction of the SIL peptide is most appropriate, though not free from bias. Mathematical modeling of this method reveals that overestimation of protein quantities would still result when rapid peptide decay occurs and that this bias would be further exaggerated by slow proteolysis. We derive a simple equation to estimate the bias for each peptide based on the relative rates of production and decay. According to this equation, nearly half of the peptides evaluated here were estimated to have quantitative errors greater than 10% and in a few cases over 100%. We conclude that the instability of peptides can often significantly bias the protein quantities measured in protein cleavage isotope dilution mass spectrometry-based assays and suggest peptide stability be made a priority when selecting peptides to use for quantification.

Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis.

The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.

Regulation of phenylalanine ammonia-lyase (PAL) gene family in wood forming tissue of Populus trichocarpa.

Phenylalanine ammonia-lyase (PAL) catalyzes the initial step of phenylpropanoid biosynthesis in plants. Five PAL genes (PtrPAL1 to 5) have been identified in Populus trichocarpa. These genes are classified into two subgroups according to their transcript sequence similarity and tissue specificity. However, the regulation of these genes and their protein functions are not well understood. In this study, enzymatic properties of each PtrPALs were characterized based on their recombinant proteins expressed in E.coli. Subcellular localizations of each PtrPALs in stem wood forming tissue were investigated and individual PtrPAL protein abundances in cytosol and membrane protein fractions were measured using protein cleavage-isotope dilution mass spectrometry (PC-IDMS). Protein/mRNA ratios of PtrPALs were further verified using RNA-Seq and gel-enhanced liquid chromatography mass spectrometry (GeLC-MS). All PtrPALs have similar catalytic properties for the deamination of L-phenylalanine, their major substrate. All PtrPALs have similar subcellular locations in stem wood forming tissue, with major amount in the cytosol (93-96 %) and less in the membrane (4-7 %). However, the protein/mRNA ratios of subgroup A (PtrPAL2, 4 and 5) are about five times that of subgroup B (PtrPAL1 and 3) in stem wood forming tissue, while all PtrPALs have similar transcript abundances. These results indicate a greater functional significance of subgroup A PtrPALs for stem wood formation, and highlight the role of gene post-transcriptional regulation.

Cheaper, faster, simpler trypsin digestion for high-throughput targeted protein quantification.

Introduction: LC-MS-based methods for protein quantification have a stigma of being relatively expensive and low-throughput. This is partly due to the cost and speed of trypsin digestion, which has primarily focused on advancements in research-based biomarker discovery applications that rely on protein/peptide identifications rather than clinical biomarker quantification. However, there is a need for simple, fast, and reproducibly efficient surrogate peptide recovery in clinical biomarker quantification. Methods: Multiple methodologies were evaluated to enhance tryptic digestion for the analysis of thyroglobulin, a prototypical serum protein biomarker. The main criteria for assessment were the yield and speed of formation of surrogate peptides. Various factors such as different additives, types of trypsin, microwave- and pressure-assisted systems, and enzyme concentration were considered as key variables, in addition to digestion time. Results: It was observed that digestion additives/denaturants had a significant impact on the speed and yield of digestion for each surrogate peptide. Increasing the concentration of trypsin alone was found to accelerate digestions appreciably for most surrogate peptides, without affecting the yield. However, the use of sequencing-grade trypsins and microwave/pressure-assisted systems did not offer significant advantages over the use of 'standard-grade' TPCK-treated trypsin in combination with a conventional incubator, once digestion time and additive had been optimized. Conclusion: We have dispelled the notion that trypsin digestion is inherently slow and expensive for targeted quantification of serum proteins. Additionally, we have established a groundwork for experimentation that can pave the way for the creation of efficient trypsin digestion protocols, aiming to optimize yield, speed, and cost. It is our hope that these advancements will promote the wider incorporation of such assays in clinical laboratories.

The pipetting Olympics: Propagating proper pipetting a priori in clinical LC-MS/MS analysis.

Introduction: Engaging pipetting events were developed to assess and challenge technicians' practical sample handling using matrices common to the clinical laboratory. As correct pipetting stands as a prerequisite for accurate clinical laboratory testing, this helped to understand sources of imprecision and bias attributed to the underlying step of aspirating and dispensing patient samples and internal standard in clinical LC-MS/MS assays while highlighting the importance for the clinical laboratory to evaluate this source of variability on an on-going basis and mitigate its impact. Methods: The events involved pipetting water, methanol, serum, and whole blood. Gravimetric analysis was used to determine the exact volumetric delivery of each matrix using two different techniques. Imprecision and bias were calculated based on the volume derived from the mass and density of each matrix, using literature values for each matrix type. Results: Low imprecision and bias were observed when pipetting water, as in common commercial pipetting assessment programs. Significantly increased imprecision and bias were observed in more applicable matrices (i.e., serum, whole blood, and methanol), indicating that water-based pipetting proficiency assessment leads to a false sense of technical ability. Additionally, the events within illuminated areas for training, leading to improved imprecision and bias. It was shown that pre-rinsing (aspirating and dispensing matrix three times to coat the tip) improved bias, particularly for delivery of methanol and whole blood. Conclusions: Precise and accurate pipetting within the clinical laboratory should not be taken for granted, nor implicitly inferred from proficiency assessment using aqueous solutions. The engaging and collegial events fostered training opportunities. Assay-specific patient sample delivery considerations (pipets and matrices) can inform the practicality of these events - the Pipetting Olympics - and drive improvements within the laboratory.

Pre-analytical conditions influencing analysis of folate in dried plasma microsamples.

Introduction: Determination of folate insufficiency is of considerable interest given its importance in fetal development and red blood cell formation; however, access to blood tests may be limited due to the requirement for phlebotomy as well as controlled temperature shipping of blood specimens to laboratories for testing due to the inherent instability of folate and its vitamers. Methods: An LC-MS/MS test was developed and validated for the measurement of 5-methyltetrahydrofolate (5MTHF) in dried plasma specimens collected from fingerstick blood using a laminar flow blood separation device, as well as liquid venous plasma for comparison. Two pre-analytical factors investigated influencing the measurement of 5MTHF in dried plasma were hemolysis of the fingerstick blood during collection and storage/shipment of the dried plasma. Results: Although observed infrequently, hemolysis >10 % resulted in elevated 5MTHF measurements, but hemolysis >1 % resulted in elevated chloride measurements, which were necessary to normalize 5MTHF measurements for variation in volume of dried plasma specimens. Stability of 5MTHF was improved in dried plasma relative to liquid plasma at ambient temperatures, but not sufficiently to allow for uncontrolled temperature shipping despite controlling for humidity and light exposure. Shipping studies emulating ISTA procedure 7D were conducted with a reusable cold packaging solution. The packaging failed to stabilize 5MTHF in dried plasma specimens during a 2-day summer shipping evaluation, but did provide sufficient temperature control to stabilize 5MTHF during the overnight shipping evaluation. Conclusion: Our studies provide boundary conditions with respect to hemolysis, storage, and shipping for successful analysis of 5MTHF from dried plasma specimens.

Comprehensive quantification of monolignol-pathway enzymes in Populus trichocarpa by protein cleavage isotope dilution mass spectrometry.

The economic value of wood/pulp from many tree species is largely dictated by the quantity and chemical properties of lignin, which is directly related to the composition and linkages of monolignols comprising the polymer. Although much is known regarding the monolignol biosynthetic pathway, our understanding is still deficient due to the lack of quantitative information at the proteomic level. We developed an assay based on protein cleavage isotope dilution mass spectrometry (PC-IDMS) for the determination of all potential, primary enzymes involved in the biosynthesis of monolignols and the peroxidases responsible for their polymerization to form lignin in the model tree species, Populus trichocarpa. Described is the identification of quantitative surrogate peptides through shotgun analysis of native and recombinant proteins, optimization of trypsin proteolysis using fractional factorial design of experiments, and development of a liquid chromatography-selected reaction monitoring method for specific detection of all targeted peptides. Of the 25 targeted enzymes, three were undetected in the normal xylem tissues, and all but two of the detectable species showed good day-to-day precision (CV < 10%). This represents the most comprehensive assay for quantification of proteins regulating monolignol biosynthesis and will lead to a better understanding of lignin formation at a systems level.

Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC-MS/MS based protein quantification and metabolic flux analysis.

Flowering plants have syringyl and guaiacyl subunits in lignin in contrast to the guaiacyl lignin in gymnosperms. The biosynthesis of syringyl subunits is initiated by coniferaldehyde 5-hydroxylase (CAld5H). In Populus trichocarpa there are two closely related CAld5H enzymes (PtrCAld5H1 and PtrCAld5H2) associated with lignin biosynthesis during wood formation. We used yeast recombinant PtrCAld5H1 and PtrCAld5H2 proteins to carry out Michaelis-Menten and inhibition kinetics with LC-MS/MS based absolute protein quantification. CAld5H, a monooxygenase, requires a cytochrome P450 reductase (CPR) as an electron donor. We cloned and expressed three P. trichocarpa CPRs in yeast and show that all are active with both CAld5Hs. The kinetic analysis shows both CAld5Hs have essentially the same biochemical functions. When both CAld5Hs are coexpressed in the same yeast membranes, the resulting enzyme activities are additive, suggesting functional redundancy and independence of these two enzymes. Simulated reaction flux based on Michaelis-Menten kinetics and inhibition kinetics confirmed the redundancy and independence. Subcellular localization of both CAld5Hs as sGFP fusion proteins expressed in P. trichocarpa differentiating xylem protoplasts indicate that they are endoplasmic reticulum resident proteins. These results imply that during wood formation, 5-hydroxylation in monolignol biosynthesis of P. trichocarpa requires the combined metabolic flux of these two CAld5Hs to maintain adequate biosynthesis of syringyl lignin. The combination of genetic analysis, absolute protein quantitation-based enzyme kinetics, homologous CPR specificity, SNP characterization, and ER localization provides a more rigorous basis for a comprehensive systems understanding of 5-hydroxylation in lignin biosynthesis.

Absolute quantification of free glutathione and cysteine in aquatic insects using isotope dilution and selected reaction monitoring.

A simple and robust isotope dilution mass spectrometry-based assay was developed for the determination of free cysteine and glutathione (GSH) in aquatic insects. Several experimental parameters were evaluated and optimized to provide specific and sensitive detection of both compounds by in situ derivatization with N-ethylmaleimide followed by acid alkylation quenching and reverse-phased liquid chromatography coupled with selected reaction monitoring. For both targets, the assay was evaluated over a concentration range of 0.313 to 320 µM and was demonstrated to have a quantitative dynamic range spanning nearly three orders of magnitude, with lower limits of quantification being 0.330 µM for GSH and 0.370 µM for cysteine. Additionally, measurements were observed to be highly reproducible over the course of several days. When applied to the analysis of four different species of insects, large biological variation between and within species was observed. Different feeding regimens were also tested within two species of insects but statistical comparisons revealed no significant difference in the levels of either compound.

Clinical irrelevance of lower titer thyroglobulin autoantibodies in patients with differentiated thyroid carcinoma.

Objective: Thyroglobulin (Tg) is an established tumor marker for differentiated thyroid carcinoma (DTC) patients. However, Tg immunoassays can be subject to Tg autoantibody (TgAb) interference resulting in incorrect Tg values. Therefore, Tg measurement with liquid chromatography-tandem mass spectrometry (LC-MS/MS) could be promising in patients with TgAbs. In this study, we compared Tg IRMA and Tg-LC-MS/MS analytically in the presence of TgAbs. Furthermore, we compared the clinical interpretation of results obtained by both Tg assays in DTC patients with lower TgAbs titers (<10 U/mL) during 131I ablation therapy. Methods: Totally 118 DTC patients diagnosed between 2006 and 2014 in a University Medical Center were followed with the Tg-IRMA (Thermo Fischer Scientific) and ARCHITECT anti-Tg (Abbott Laboratories) assays. We re-analyzed their samples with a sensitive Tg-LC-MS/MS method (Labcorp, limit of quantification of 0.02 ng/mL). Passing-Bablok regression analysis was performed on samples obtained during 131I ablation therapy and follow-up. Results: In 304 samples with lower TgAb titers, a good analytical agreement was found between both Tg assays (slope of 1.09 (95% CI: 1.05-1.16)). Fifty-five samples with potentially interfering TgAbs showed higher Tg-LC-MS/MS values than Tg-IRMA (slope of 1.45 (95% CI: 1.12->>100)). In patients(n = 91) with lower TgAb titers at the time of 131I ablation therapy, the Tg assays showed a clinical concordance of 91.2, 87.9, and 98.9%, respectively, using a Tg cut-off value of 1.0, 2.0, and 5.0 ng/mL. Conclusions: In DTC patients with lower titer TgAbs, Tg-IRMA is still a reliable and useful tumor marker. In DTC patients with potentially interfering TgAbs, Tg-IRMA values decreased due to TgAb interference.

Capitalizing on the hydrophobic bias of electrospray ionization through chemical modification in mass spectrometry-based proteomics.

Protein chemical derivatization has emerged as an invaluable bioanalytical approach in mass spectrometry-based proteomics with nearly unlimited potential. To date, derivatization strategies in proteomics have primarily focused on improving mass spectral identification and relative quantification of proteins, as well as increasing enrichment yield from complex mixtures. However, there is a great opportunity to develop and exploit front-end chemical processes to enhance the ability to detect low-abundant peptides and proteins for a large number of applications. The content of this article focuses on improvements in targeted, mass spectrometry-based proteomic strategies, achieved by taking advantage of the mechanism of ESI through the use of hydrophobic chemical derivatization.

Utilizing spectral counting to quantitatively characterize tandem removal of abundant proteins (TRAP) in human plasma.

Biomarker discovery efforts in serum and plasma are greatly hindered by the presence of high abundance proteins that prevent the detection and quantification of less abundant, yet biologically significant, proteins. The most common method for addressing this problem is to specifically remove the few abundant proteins through immunoaffinity depletion/subtraction. Herein, we improved upon this method by utilizing multiple depletion columns in series, so as to increase the efficiency of the abundant protein removal and augment the detection/identification of less abundant plasma proteins. Spectral counting was utilized to make quantitative comparisons between undepleted plasma, plasma depleted with a single depletion column, and plasma depleted using two or three depletion columns in tandem. In the undepleted plasma only 29 lower abundance protein groups were identified with the top-scoring protein from each group having a median spectral count of 3, while in the plasma processed using a single HSA depletion column 61 such protein groups were identified with a median spectral count of 8. In comparison, 76 lesser abundant protein groups were identified with a median spectral count of 11.5 in the two column setup (i.e., HSA followed by MARS Hu14). However, in the ultimate depleted plasma sample, which was created using three depletion columns in tandem, the number of less abundant protein groups identified increase to 81 and the median spectral count for the top-scoring proteins from each group increased to 15 counts per protein. Moreover, exogenous B-type natriuretic peptide-32, which was added to the plasma as a detection benchmark at 12 µg/mL, was only detected in the plasma sample depleted using three depletion columns in tandem. Collectively, these data demonstrate that this method, tandem removal of abundant proteins or TRAP, provides superior removal efficiency compared to traditional applications and improves the depth of proteome coverage in plasma.