Mass-Linked Immuno-Selective Assays in Targeted Proteomics

Review pubs.acs.org/ac

Mass-Linked Immuno-Selective Assays in Targeted Proteomics Ashraf G. Madian,† Nishi S. Rochelle,† and Fred E. Regnier*

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Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States

CONTENTS

Mass-Linked Immuno-Selective Assay (MALISA) Modes of Analysis Top-Down MALISA Bottom-Up MALISA Mixed-Mode MALISA Applications of MALISA Type 1 MALISA Type 2 MALISA Type 3 Assays Type 4 Assays Conclusions Author Information Corresponding Author Author Contributions Notes Biographies References

these problems. On the basis of more than 50 years of experience with radio immunological-assay (RIA)7 and enzyme-linked immunosorbent-assay (ELISA)8 methods, it has been shown that antibody-directed analyte selection rapidly reduces sample complexity at minimal cost, with a high degree of structural specificity.9−11 Immune-specific selection in the past, and probably long into the future, will play a major role in targeted protein analysis but in a different way than in RIA and ELISA. A problem in immune-specific selection of proteins as a means to assess biological activity is that an antibody may target a different component of structure than the feature(s) determining biological activity.12,13 In the past, it has always been assumed that the two are directly related, but that may not be true. Proteins can exist in a series of variant forms, differing in the type, number, and location of differentiating features within their primary, secondary, tertiary, and quaternary structure. Posttranslational modifications, splicing variations, conformers, and mutation-based amino acid substitutions are the most frequent isoform differentiators. It is easily seen how an antibody targeting a specific feature of a protein, or set thereof, could coselect isoforms not related to its biological activity, as is probably the case in assays of prostatespecific antigen14 for example. Unfortunately MS-assisted immunological assays are described in so many ways that they are difficult to trace in the literature. Reports on mass spectrometric immunoassays (MSIA),15 affinity-MS, probe affinity mass spectrometry (PAMS),16 immunoMALDI (iMALDI),17 immunochromatographic-mass spectral analysis, surface-enhanced laser desorption ionization-TOF (SELDI-TOF),18 and surface-enhanced affinity capture (SEAC) with SELDI-TOF19 are very similar assay methods, focusing on protein level detection by MS. Similarly, there are assay methods for protein digests, described as affinity peptidomics,20 bottom-up affinity proteomics methods,21 iMALDI,17 and stable isotope capture with antipeptide antibodies (SISCAPA),22 that employ essentially the same analytical methodology but with a still different terminology. This is confusing. Clearly, the nomenclature used to designate mass spectrometry-assisted immunological assays might be unified and simplified. We suggest that mass-linked immuno-selective assay (MALISA), an eponym of ELISA, is an easier way to compare MS-assisted immunological-assay methods, reflecting the origin, similarity, and also the difference between these immunological-assay technologies. This term is neither meant to replace existing terms nor meant to be standard nomenclature for describing all future methods. MALISA is used here as a means to unify the discussion of mass spectrometry-linked immunological-

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he issue of how to rapidly identify and quantify multiple proteins of known structure in biological samples is a subject of great interest today, particularly in the biomarker validation and clinical proteomics arenas. The potential of mass spectrometry (MS) in this endeavor is an important issue.1−4 Will MS-based methods be superior to current protein-assay technology for routine monitoring? Does MS have sufficient sensitivity and accuracy for clinical use? Can it compete with conventional assays in cost, speed, ease of use, and reliability? In short, what role will MS play in the future of routine protein assays? The ease and speed with which peptides can be resolved, fragmented, sequenced, identified, and quantified in the gas phase5 is an extremely valuable asset of MS, as we have learned from proteomics.6 But as a prelude, proteins in samples must undergo purification and enrichment along with reduction, alkylation, and proteolysis in many cases before they are ready for MS analysis. Whereas MS analysis occurs in milliseconds or less, much of the preliminary sample preparation is achieved in lengthy manual or robotic steps with substantial variability and expenditure of time. Although tolerable in discovery, extensive sample manipulation, deficiencies in selectivity, and poor reproducibility compromise MS detection strategies in routine analysis. Sample complexity and the need for preliminary fractionation are major issues in proteomics, especially when the number of components exceeds the analytical limit of a mass spectrometer. Suppression of ionization, the need for high resolution, reduction of dynamic range, and diminished confidence levels in identification and quantification are all byproducts of highly complex samples. Reduction in complexity is the best solution to © 2012 American Chemical Society

Special Issue: Fundamental and Applied Reviews in Analytical Chemistry Published: September 5, 2012 737

dx.doi.org/10.1021/ac302071k | Anal. Chem. 2013, 85, 737−748

Analytical Chemistry

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down MALISA method has been very effective in identifying protein variants of clinical significance30 but is dependent on the ability to launch intact antigens into the gas phase. A second MALISA strategy uses the bottom-up protocol described by Hunt26 (Figure 1) for identification of proteins in which samples are trypsin-digested before antibody capture and LC-MS/MS identification. Although the intent is still protein identification, fractionation and identification are achieved exclusively at the peptide level, as in “shotgun” proteomics.31 When possible, multiple signature peptides unique to the antigen are selected with different polyclonal antibodies.32 A third form, the mixed-mode approach, involves a combination of the top-down and bottom-up strategies. Antigens are affinity-selected (and sometimes further fractionated) at the protein level after which they are digested and the peptide fragments identified by RPC and tandem MS, as initially described by Hsieh (Figure 1). The mixed-mode and top-down MALISA methods have the advantage that a single antibody is used to select and identify a protein, while the bottom-up and mixed-mode approaches can accommodate any size antigen. Table 1 lists the different characteristics, advantages, and disadvantages of the three modes. As was the case with proteomics, it is seen (Figure 1) that the use of MS-assisted immunological assays was enabled by a series of innovations in separation science and mass spectrometry. Clearly these advances have enabled life scientists and immunologists to create new types of immunological assays (Figure 2) that were impossible with the old ELISA and RIA technologies. Those new types of assays will be described in the Applications of MALISA. Top-Down MALISA. Each of the MALISA modes described below arose in a different way, often for a different reason. The beauty of top-down MALISA is its simplicity. It takes the early work of the RIA and ELISA community and says “there is a better way to detect antigens captured by immobilized antibodies, that is, by determining the mass of captured antigens”. Antigen isoforms can often be recognized, and throughput can be high; single sample analysis time is equivalent to that of an ELISA, and multiplexing is possible. Top-down MALISA has been achieved almost exclusively with MALDI-MS. The method arose shortly after the description of MALDI by Tanaka33 and the pioneering work of Karas and Hillenkamp34 in 1988, along with Hunt’s report of bottom-up MALISA in 1991 (Figure 1).26 Top-down MALISA was first described by Papac et al. in 1994 with their observation that cytochrome c could be recovered from a monoclonal antibody (mAb) affinity chromatography column and the molecular weight determined to within 0.7% by MALDI-MS.27 The surface-enhanced laser desorption ionization (SELDI) mass spectral analysis35 method developed by the Hutchens group a year earlier is similar, but it used DNA instead of antibodies to capture lactoferrin from preterm infant urine.36 True MALISA versions of SELDI based on surface-enhanced affinity capture (SEAC) using antibodies to select antigens at the MALDI probe came later.19 Most of the top-down MALISA methods use a common set of steps (Figure 3), clustering around antigen−antibody complex formation on a surface, subsequent release of antigens from the immune complex, and their direct identification by MALDI-MS. There are generally no additional fractionation steps beyond immune complex formation. Antibodies in top-down MALISA have been immobilized individually,37 in a mixture, or as arrays of individual antibodies38 by either covalent immobilization39

assay methods that are in current use or projected to be employed in the future. Moreover, it will be shown that the various MALISA protocols are simply specialized forms of targeted protein analysis that can be further differentiated by the terms “top-down”, “mixed-mode”, and “bottom-up” used widely in proteomics to describe major analytical strategies.23,24 This does not mean, however, that MALISA is synonymous with proteomics. When small numbers of antigens are being selected, it is not. MALISA can even be considered as a way to avoid proteomics. This review will be divided into two parts, one dealing with the origin and current status of the various Mass-Linked ImmunoSelective Assay (MALISA) Modes of Analysis, and another on Applications of MALISA. Some modes of MS-assisted immunological assays will not be discussed. These are methods in which (i) the antigen, antigen fragments, and a derivative thereof are not detected directly by MS, (ii) lanthanide-tagged antibodies are used,25 (iii) two-dimensional (2D) gel electrophoresis methods are used, and (iv) MALISAs of nonpeptide haptens are used. The objectives of this review are to address how MS is being linked to antibody selection of antigens, compare the methods of linking, and describe how the type of linkage enables new types of immunological assays that go beyond ELISA. A discussion on the many biological problems that have been examined with these MS-based immunological assays is beyond the scope of this review. For example, in the case of SELDI, the method itself will be discussed but not the hundreds of papers where the technique is applied.

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MASS-LINKED IMMUNO-SELECTIVE ASSAY (MALISA) MODES OF ANALYSIS Mass-assisted immunological assays were first described by the Hunt laboratory in 1991 (Figure 1). Having recognized that

Figure 1. Chronological evolution of technologies that enabled the various MALISA modes. The most important were the matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) modes of mass spectrometry.

peptide antigens captured on immunosorbents could be released and examined by MS, they proceeded to demonstrate mass spectral analysis as an alternative to radiolabeling and enzymatic amplification as a means of detecting peptides selected in immunological assays.26 Multiple forms of MALISA have since been described based on differences in the coupling steps used to construct an assay (Figure 2). One form is a top-down approach in which antigens are captured with an antibody and examined directly by MS without modification. As seen in Figure 1, this mode of MALISA originated with seminal work from the Papac,27 Nelson,28 and Hutchens29 laboratories ca. 1993−1998, immediately following introduction of matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This top738



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Figure 2. Modes of MALISA. “D” stands for dimension. Specificity increases with the number of analytical dimensions by eliminating interfering species through more stringent fractionation. Differentiators in MALISA are whether (i) antigen capture with an antibody is occurring at the protein or peptide level (yellow), (ii) detection is occurring at the intact protein or peptide level (red), or (iii) chromatographic means are being used to achieve additional fractionation (purple). Top-down MALISA has the fewest dimensions of discrimination. The point at which proteolysis occurs (brown) is another difference. The order in which unit operations are coupled is still another differentiator. Although the same set of unit operations are used in bottom-up and mixed-mode MALISA, they have different capabilities based on the order in which the operations are coupled.

(Figure 3A), direct adsorption40 (Figure 3B), or affinity capture with an immobilized protein41,42 such as protein G (Figure 3C), all with equivalent efficacy. There are, however, several points worth noting. One is the importance of nonspecific binding of proteins and peptides by the solid-phase sorbent. Because there is no further chromatographic fractionation in top-down MALISA beyond immune-specific selection on the solid-phase sorbent, nonspecifically bound proteins and peptides are likely to be desorbed along with antigen(s) and increase the background in mass spectra. This diminishes both selectivity and the detection limit. A second issue is the importance of removing the sorbent matrix before detection. Sorbent matrices can diminish ionization in MALDI-MS and when possible should be removed. Antigen release and transport to the MALDI plate in steps 2 and 3 (Figure 3) can be achieved in either order with similar outcomes.43,44 MALDI matrix can be added either during antigen−antibody dissociation or afterward. When done together, the number of steps is reduced. Execution of the steps outlined in Figure 3 has been accomplished in multiple ways that are quite different. One is through some type of chip approach in which antibodies are immobilized on the planar surface of the chip,19 sequestered in a gel matrix on the chip surface,45 or bound in a pore matrix etched into the chip surface.39 Generally, the specific surface area of a planar chip is so low that the loading capacity and linear dynamic range are limited. Surface plasmon resonance (SPR) chips are much better in that a gel matrix is frequently attached to the chip surface to increase loading capacity.41,46−50 Another advantage of immune selection on an SPR chip51−57 is that antigens are

quantified in a second way, independent of MS. Antigens thus captured are then desorbed from the SPR chip, deposited on a MALDI probe, and detected again by MALDI-MS.28,58 This double-detection strategy has the advantages of further confirming the MALDI-MS results, detection of cross-reacting species that could have contributed to the SPR signal, and detection of antigen variants differing in mass. Multiplexing has been an important advance in tandem SPR-MS,59 having been achieved with multiple flow cells in which one to two proteins are detected per flow cell or in a single flow cell with up to six arrayed antibodies.60,61 The detection limit of antibodies immobilized on 200−750 μm diameter array elements was determined to be approximately 1 nM.62−64 Anodic etching of silicon is another way to increase the surface area of planar chip surfaces.39 This process allows production of arrays with macroporous surface elements in which the pores are a few nanometers to ∼100 nm in depth and bear surface area enhancements of 100−1000-fold, depending on the etching time and voltage. Antibodies are dispensed in picoliter droplets onto array elements using a piezoelectric printer and adsorbed directly65 (Figure 3B). Samples can then be autodispensed onto array elements where they are constrained as 5−15 μL droplets above array elements by a 200 μm-wide silicon dioxide ring around the array. Subsequent to antigen capture, sample zones were washed multiple times and the antigens released with an acidic denaturing agent. Following antigen desorption and aspiration from the surface by a sample-handling robot, aliquots were deposited into microfabricated 48 nL vials containing 50 μm of POROS R2 reversed-phase particles. The vials are of an inverted pyramid structure with 54.74° angle walls, a 15 μm 739



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Table 1. Characteristics, Advantages, and Disadvantages of Different Modes of MALISA MALISA mode

number of dimensions

elapsed time

top down

two

15−30 min

throughput 100/h

quantitation -use of an internal standard is recommended

general advantages

general disadvantages

-can recognize protein families

-efficiency by which large antigens are transferred in the gas phase is an issue -poor quantitation -the degree to which multiplexing is possible with comparable ionization efficiency is a concern -difficulty in locating multiple unknown variations within the antigen structure -no sequence data are produced -limited resolution of impurities from antigens before MS analysis

-simple -inexpensive

-analysis time is equivalent to that of ELISA -multiplexing is possible -retains post-translational modification (PTM) and cleavage information -high-throughput

bottom up

four

24 h

2/h

-MRM -SRMa -label-free (only signature peptides can be used for quantitation)

-excellent quantitation and identification -best sensitivity -multiplexing is possible -detection limits in the low ng/ mL range

mixed mode

five

30 min

0.5−2 /h

-MRM

-can recognize protein families and isoforms -simple

-SRM -label-free (both signature and nonsignature peptides can be used for quantitation)

-multiplexing is possible

-larger proteins are not identified as easily due to size bias -limited loading capacity on MALDI probe surfaces -differentiation between isoforms of the same mass is problematic -cannot recognize protein families -low-throughput -loss of PTM and cleavage information -destruction of functionally relevant structures -a different antibody will be required for each structural feature being targeted -identical peptides of different protein origins will coelute -linear dynamic range -the efficiency with which peptides critical to isoform differentiation is seen -peptide recovery is dependent on the immobilized enzyme reactor (IMER)

-automation increases throughput -analysis time is equivalent to that of ELISA a

SRM is selective reaction monitoring.

through-hole at the bottom with a depth of 310 μm, and a 466 × 466 μm square opening at the top. Ninety-six vials were arrayed in the format of a microtiter plate to allow the use of standard immunological-assay robots for sample and solvent dispensing. This configuration allows 10 μL portions of sample along with wash solvents to be drawn through the POROS particle bed in all 96 vials simultaneously by application of a vacuum to the back of the plate at the 15 μm openings in the bottom of vials. Following the washing steps, proteins or peptides were eluted from the POROS beads with an acetonitrile-containing MALDI matrix. During the course of elution from the bottom of the vials, effluent accumulates as a drop on the back of the chip at each vial opening and evaporates, leaving behind a crystalline MALDI matrix with antigens on the plate surface. The dried chip is then inverted and inserted into a MALDI-MS instrument for analysis. The detection limit in the case of angiotensin I was 10 fmol, starting with a diluted plasma sample (10 μL, 1 nM). Sample throughput in devices of this type will be large while maintaining a small footprint. Still another approach to solid-phase extraction is through the use of immunosorbents on magnetic nanoparticles.66 The advantage of magnetic nanoparticles over conventional magnetic particles is that they are of higher surface area per unit mass and

of enhanced loading capacity. Subsequent to antigen capture and washing, the nanoparticles are transferred to a MALDI target where antigens are released and examined by MALDI-MS. Particles left on the MALDI target do not compromise MALDI detection, according to the authors. Probe affinity mass spectrometry (PAMS) and surfaceenhanced affinity capture (SEAC) go one step further; antigen capture is carried out on the MALDI target directly.16,67,68 There is no transfer from a sensor chip or column to the MALDI target. The antibody is immobilized on the probe surface either directly or by coupling to some organic matrix on the probe. As noted above, the advantage of using a gel matrix on probes is that it allows greater antibody loading capacity but has the accompanying concern that ionization efficiency could be compromised by the residual matrix. These methods reduce sample manipulation and reportedly increase sample enrichment several hundredfold by minimizing dilution and allowing higher antibody loading through the use of gel matrices. Multiplexing and multiple levels of analyte fractionation have been achieved with the SEAC version of SELDI-TOF in both discovery and routine analysis.69,70 Biomarker candidates have been discovered and evaluated in the cases of breast, lung, prostate, colorectal, and ovarian cancer, along with Alzheimer’s disease, using SELDI740



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Figure 3. Illustration of the basic solid-phase extraction strategies that have been used in top-down MALISA. The diagram in panel A shows how antibodies covalently immobilized on a solid-phase support can be used to capture antigens from a sample, and after removal of unbound proteins by washing, antigens are released for analysis by MALDI-MS. Although not shown, sorbent removal is desirable to preclude reduced sensitivity. Panel B illustrates immobilization by direct adsorption of antibodies on a polysilicon surface. During the desorption step, immobilized antibodies may be desorbed along with the antigen. It is intended in this case that the solid-phase sorbent will be used once and then discarded. Panel C illustrates the use of alternative solid-phase extraction strategies for capturing immune complexes. A solid-phase binding agent (Spb) such as protein A or G is immobilized in this case and used to capture either the antibodies or an immune complex.

TOF.71−76 Many of the SELDI papers focus on the unique way informatics was used in discovery, but that is beyond the scope of this review. The focus of this review is on how assays are executed and not on the applications of the method. A fiber-optic probe based on surface plasmon resonance (SPR) detection with a polyclonal antibody (pAb) on the surface of the fiber-optic probe77 goes beyond PAMS and SEAC in integrating detection modes, but again there is concern about matrices interfering with ionization. Without an independent optical method of quantification, there is concern regarding quantification of all the top-down methods. The addition of an internal standard derivative of the antigen (ISDA) prior to MALDI-MS78,79 would overcome variability issues but has not been widely employed to date. An ISDA deals with many of the common problems in MALDI-MS quantification ranging from (i) heterogeneity of analyte distribution across matrix crystals and (ii) variations in signalto-noise ratio across the target surface to (iii) shot-to-shot variability of the laser and (iv) matrix suppression of ionization. Using a 13C-labeled isotopomer of the antigen as an internal standard allowed detection limits of ∼15 pM with a linear dynamic range of at least 2 orders of magnitude80 and a throughput of ∼50−100 samples/h.81 This is comparable to ESIMS methods in sensitivity while being simple, fast, accurate, reliable, versatile, low cost, and quantitative. Other concerns with top-down MALISA are (i) the efficiency with which large antigens and protein complexes can be transported into the gas phase, (ii) locating multiple unknown variations within an antigen, (iii) differentiation between isoforms of the same mass, (iv) limited loading capacity on MALDI probe surfaces, (v) the degree to which multiplexing is

possible with comparable analyte ionization efficiency between analytes, (vi) limited resolution of impurities from antigens before MS, and (vii) the fact that no sequence data are produced. Bottom-up and mixed-mode MALISA, in contrast, use 4−5 orthogonal dimensions of analysis and provide a peptide sequence. Finally, the method has a size-bias impediment in the current form. Large proteins are not identified as easily. These issues will perhaps be resolved in future instrumentation but are a concern today. Bottom-Up MALISA. Immunological assays have been achieved with protein-targeting antibodies for half a century. The most differentiating feature of bottom-up MALISA is that this old strategy was abandoned in favor of selecting peptide fragments of proteins from tryptic digests. What would be the motivation for digesting proteins to generate a mixture with at least 50-fold more components than the initial sample, destroying valuable structural information during proteolysis, converting proteins into peptides that are less immunogenic, and using multiple antibodies to achieve an assay instead of one, as is the case with other forms of MALISA? It can be argued82 that “quantification is easier with ESI-MS analysis of peptides than with MALDI-MS or ESI-MS of proteins, antibodies can be produced using simple synthetic peptides conjugated to immunogens instead of protein antigens that are difficult to isolate (or may never have been isolated), the requisite ionization energies for MS analysis can either be worked out with synthetic peptide standards or predicted in silico, sensitivity of peptide detection is superior with high duty cycle ESI-MS instruments, and multiplexing is easily achieved”. These two sets of arguments are very different. Both have merit; the later is based primarily on facilitating MS detection, while the former focuses more on 741



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appear to suffer from nontargeted peptide binding. Peptide identification was achieved by standard RPC-MS/MS.102,102,103 Enrichment ranged from 1800 to 18000-fold with parent protein detection limits of 1 ng/mL. Some of the limitations and issues surrounding bottom-up MALISA have been noted above, the major one being the destruction of valuable structural information during the initial proteolysis of samples. This precludes selection of protein families and proteins that differ in their tertiary or quaternary structure. Even post-translational modification variants will be hard to identify. Also, a different antibody will be required for each structural feature being targeted. Some peptides from a protein will be common to all isoforms, others common to a few of the isoforms, while still others isoform-specific. The need for signature peptides at the isoform level is apparent. Finally, there is the issue of identical or cross-reacting peptides from other proteins in the proteome. This problem will be addressed at greater length below. Mixed-Mode MALISA. This mode of MALISA is a hybrid of the top-down and bottom-up methods. The strength of this method is that “either single gene or in many cases multiple gene expression products and their isoforms can be captured by a single antibody with full retention of native structural information, the protein species thus chosen can be further fractionated, and the selected protein fraction(s) converted to a peptide mixture for RPC-MS/MS identification and quantification of peptides common to one or all isoforms of the parent proteins”. This form of MALISA facilitates recognition and quantification of either differences or similarities in polypeptides selected by an antibody, changes in expression within a protein family, post-translational modifications, ratios of expression versus post-translational modifications (PTMs), single-amino acid polymorphism, and identification of splice variations. Although the primary strength of the method is in the analysis of known proteins, it can also be used in discovery, particularly in the case of targeting groups of proteins that carry the same posttranslational modification or cross-reacting epitopes. Mixed-mode MALISA arose from immunochromatographic analysis (ICA),12 a liquid-chromatographic detection method used in immunological assays. With incorporation of ESI-MS detection and addition of an immobilized enzyme reactor (IMER) for online trypsin digestion, the first automated LC-MS/ MS system capable of mixed-mode MALISA104 emerged in 1996. Further inclusion of an automated reagent-dispensing system allowed reduction, alkylation, and immune-complex formation in sample vials before introduction into the RPC-MS/MS system.105 Immune complexes formed in solution are selected from samples using a gigaporous protein G or avidin affinity sorbent to maximize adsorption kinetics, subjected to a 50−100 volume wash to remove weakly bound proteins, and desorbed from the affinity sorbent with an acidic mobile phase; selected proteins are digested with a trypsin IMER and the resulting peptides desalted and concentrated on a polystyrene-divinylbenzene particle matrix, and the enriched peptide mixture is examined by LC-MS/MS. With an immobilized polyclonal antibody, this approach has been used to select normal and structurally aberrant hemoglobins with common structural domains that, subsequent to proteolysis, were differentiated by their signature peptides in ∼30 min through RPC-MS/MS.107 Quantification in mixed-mode MALISA has been achieved at the peptide level using an in vitro derivatizing agent such as iTRAQ for relative quantification,106−108 a 13C-labeled internal standard peptide for absolute quantification in the MRM

biological issues. The great advantage of bottom-up MALISA is its similarity to shotgun proteomics. So much of the MS instrumentation available today has been designed for bottom-up proteomics that this is an advantage. As noted above, the Hunt laboratory first described bottom-up MALISA in 1991.31 Recognizing that both free and conjugated tryptic peptides elicit antibody synthesis,83 and that peptides captured by antibodies should be identifiable by mass spectrometry,84 they developed and launched an immunespecific bottom-up method for detecting tryptic peptides of proteins using FAB-MS. With the subsequent discovery of ESIMS,85 multiple-reaction monitoring,86−88 and isotope coding of synthetic peptides,89 bottom-up MALISA has evolved in many directions, including epitope mapping,90,91 identifying protein− protein interactions, affinity peptidomics,92,93 and quantification by stable isotope standards and capture by antipeptide antibodies (SISCAPA).22 Antibody affinity plays an important role in the resulting sensitivity of antibody-based assays of peptides. Because peptides are far less immunogenic than proteins, if they elicit antibody synthesis at all, they are generally conjugated to a carrier immunogen for antibody production. Ideally, one would like to have high-affinity antibodies against the peptide targets, but it has been found that both conjugation ratios and the molecular orientation of peptides in conjugates have a major effect on pAb titers.94 This greatly complicates mass production of the highestaffinity antibodies. The complication with low-affinity antibodies is that peptides will partially elute during extensive washing of immune complexes, and antigen capture will no longer be quantitative. Nonetheless, a multiplexed immunization strategy has recently been described in which up to five peptides from each of 89 proteins (i.e., 445 peptides) were used together to individually immunize rabbits.28 Of the 220 antibody candidates evaluated, working assays were developed for all 89 proteins. Peptides were detected with more than half the proteins at 0.5 pmol/mL (i.e., approximately 100 ng/mL parent protein) based on quantification by multiple-reaction monitoring. The possibility that higher affinity and specificity can be obtained with monoclonal antibodies (mAb) is under investigation.95 Quantification in all forms of proteomics is widely achieved by multiple-reaction monitoring (MRM).96 Although relatively new to proteomics, MRM-based quantification in complex mixtures of small molecules was originally described in 1978 by the Cooks group.84 This method has been applied to peptide quantification by the addition of stable isotope-coded peptide standards to samples at known concentrations either before or after trypsin digestion, but before LC-MS/MS analysis, with the assumption that proteins are reduced to limit tryptic peptides. The isotope ratio of a limit tryptic peptide to a labeled peptide standard is then used to calculate the absolute concentration of protein parents in samples, assuming a 1:1 concentration ratio between the limit tryptic peptide and parent protein. It is important to note that this assumption is not always true due to incomplete proteolysis. With bottom-up MALISA, peptide standards may be added either before or after capture and enrichment. The special case where standards are added after proteolysis but before peptide capture has been called SISCAPA.97−99 This method has undergone several stages of evolution,95,100,101 the most recent iteration being the use of immobilized antibodies on magnetic beads to capture tryptic peptides and internal standards.28 After suitable washing, affinity-selected peptides were released from the magnetic beads, albeit with nonspecifically bound, nontargeted peptide contamination. But nonspecific binding is not unique to magnetic beads. All solid-phase extraction systems 742



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Figure 4. Classification of MALISA applications according to the type of structural feature targeted. The abbreviations td-, mm-, and bu- signify the topdown, mixed-mode, and bottom-up modes of MALISA, respectively.

mode,109 or some type of direct quantification based on selective reaction monitoring (SRM) or spectral counting.110 These are the same methods used in many forms of proteomics. Coefficients of variation have been reported to range from 5 to 8% with the stable isotope-based internal standard methods versus approximately 15−20% with spectral counting. Clearly, internal standard methods allow superior quantification. Mixed-mode MALISA is a serial processing method in which the rate-limiting step is the RPC separation. Similar to bottom-up MALISA, this restricts throughput from 0.5 to 2 samples per hour as opposed to top-down MALISA where throughput is at least an order of magnitude higher. This derives from having fewer dimensions of discrimination and less confidence in identification in the top-down mode. Throughput is important in large-scale validation studies where large numbers of samples are being examined but is not so important in routine clinical use. Another limitation of the mixed-mode MALISA is that antibodies against rare and low-abundance proteins are harder to prepare than for the bottom-up method where synthetic peptides can be used in immunization. However, recent efforts to prepare antibodies against continuous epitopes of proteins discussed below may diminish the necessity to have native proteins as immunogens. Finally, there is the concern with nonspecific binding in the affinity capture and IMER steps.

antibodies enable new types of immunological assays not commonly seen in the past. A scheme (Figure 4) is described below that defines at least four types of assays that are greatly enhanced by detection with MS. This scheme is based on the fact that MALISA methods have two attributes: (i) the type of antigen feature targeted and (ii) the manner in which the steps are linked to achieve an analysis (Figure 2). With the use of this (Type)(Mode)-MALISA naming scheme, a type 1 assay carried out by top-down MALISA will be described as a type 1 td-MALISA. It will be shown below that, theoretically, there can be at least 12 forms of MALISA based on 4 types of antigen targeting and 3 modes of analysis. However, some forms are of limited utility. Type 1 MALISA. This type of MALISA is most like the traditional ELISA. The immune capture component of a type 1 assay is being defined here as “selection of the expression product of a single gene by targeting an epitope or epitopes in the primary, secondary, or tertiary structure of the expressed protein using either a single pAb or mAb”. Generally, all isoforms bearing a PTM, single-amino acid polymorphism, splice variation, or noncovalently bound protein partner will be selected together unless the modification has destroyed or altered the structure of an epitope critical for recognizing and binding the isoform. Differentiation between the captured species is achieved by additional chromatographic and MS dimensions. Although discrimination by molecular weight alone has been widely described with the top-down approach to biomarker screening,111−113 phenotyping,114,115 assessing diversity,116−119 and variant analysis,120−124 the addition of RPC-MS/MS in a type 1 mm-MALISA will theoretically give it greater power to recognize unique peptides from all forms of the antigen along with sites of variation. The caveat is that peptides critical to the recognition of isoforms must ionize well in the sample being examined. This may not be the case and is a weakness of all MALISA, in fact, all MS-based analysis of proteins. This is especially critical when

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APPLICATIONS OF MALISA The sections above described the various modes of executing MALISA, focusing on categorizing and describing the many protocols used to analyze and detect antigens by MS. This section, in contrast, examines discrimination among the antigens being determined, the origin of these antigens, and the structural features being probed. Antibodies vary widely in the structural features they target. Some select polypeptide features in the primary, secondary, or tertiary structure of a protein, while others distinguish between post-translational modifications. When paired with mass-spectral detection, these different classes of 743



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been identified in this manner with functions ranging from cell signaling, motility, and transport to RNA processing and protein processing. Tyrosine is also modified by nitration. Immunoprecipitation based on an anti-3-nitro Y-antibody was used with Arabidopsis thaliana in the identification of 127 proteins that putatively carry this modification.141 Being able to select proteins bearing a particular PTM or a PTM on a particular sequence is an enormous asset. Targeting antibodies N-acetyllysine142 and Nmethyllysine143 are similarly diverse in being able to target a unique PTM, the PTM in a unique sequence, or a group of lysine modifications. Methylation occurs on arginine residues as well. With lysine, the degree of methylation varies from one to three. With the use of antibodies that target lysine-methylated residues, 59 methylation sites have been identified in proteins by RPCMS/MS. 145 Arginine methylation in contrast to lysine methylation results in the formation of either asymmetric or symmetrical dimethylation of arginine residues located in RGrich clusters. Dimethyl arginine-specific antibodies have been used in the identification of ∼200 proteins that are putatively arginine-methylated.144 Major protein complexes identified in this way include components required for pre-mRNA splicing, polyadenylation, transcription, signal transduction, and cytoskeleton and DNA repair. An asset of type 2 MALISA is that the requisite antibodies are often commercially available, and multiplexing is possible with a single antibody. This means that MALISA can be used in discovery proteomics. With acetyllysine for example, 50−85 proteins of broad biological function were selected from lysates, depending on the organism.145,146 Antibody selection of glycoproteins is similar. Selection of Lewis x or sialyl-Lewis x antigen-bearing glycoproteins from cancer patient plasma samples with a single antibody generally yields mixtures of 50− 100 proteins, depending on the type of cancer.109,147 It is relatively easy to identify this number of proteins with most MS/ MS instruments, but detection limits seldom go below 1 ng/mL. The number of proteins identified increases in proportion to sample size in our experience, suggesting that low-abundance proteins are being missed. As instrument sensitivity increases, the number of proteins identified and the degree of multiplexing will increase accordingly. A limitation of type 2 MALISA is that PTM-modified peptides can be large and difficult to ionize, as we know from glycopeptides and those bearing large numbers of phosphate residues, although the proteins are easily identified through their unmodified peptides. Still another limitation is that the selectivity of PTM-targeting antibodies is sometimes not very good. False negatives and false positives have been reported.145 Type 3 Assays. Cross-reactivity is a major issue in antibodybased proteomics,148 autoimmune diseases,149 molecular mimicry,150 and allergenicity,151 all stemming from the necessity to have antibodies of high binding specificity, the ability to recognize similarity between immunogenic proteins, or an understanding of the relationship between environmental proteins and the onset of autoimmune diseases. Type 3 MALISA can perhaps play a major role in addressing these issues. The function of immune selection in this type of assay is defined as “assessing the potential that multiple gene expression products or peptides thereof from a single or from multiple organisms are being selected by a single mAb or pAb”. The objective here is to assess cross-reactivity. Because MALISA methods analyze the structure of captured antigens, they are particularly powerful in examining cross-reactivity. Evidence of cross-reactivity will be revealed by the presence of nonepitope portions of untargeted

only one peptide in a protein must be recognized, as in the case of a single amino acid variation. Multiplexing in the MALISA mode is still in its infancy. Although we know from proteomics that identifying 100 proteins in a single analysis is trivial, targeting that many proteins in an integrated immunological assay will be much more complicated. First, there is the issue of obtaining large numbers of antibodies, qualifying them, putting them into the requisite format for the assay, and validating the final multiplexed test. Evidence of the heroic effort involved in large-scale multiplexing is found in the recent work of the Anderson, Carr, and Paulovich laboratories with their development of multiplexed SISCAPA assays for the Clinical Proteomic Technology Assessment For Cancer (CPTAC) project of NCI.22,28,125 Their work on multiplexed antibody production is particularly significant in showing how the requisite antibodies for large-scale multiplexing might be prepared. On the basis of this work, we conclude that large-scale multiplexing is most justifiable in cases where the antigens have been validated to be of clinical utility and there is a long-term need for the assays, validated methods and reagents are being used in assay development, and there is some type of support system to manufacture the requisite reagents in large-scale studies. The difficulty and cost of developing clinically validated assays that meet FDA approval are likely to increase exponentially with the degree of multiplexing. Protein−protein association is widely studied with “antibody pull-down” or “immunoprecipitation” assays, especially using MS to identify binding partners.126−128 This special kind of type 1 assay exploits the fact that proteins associated with antigens in a complex are co-selected and can be identified along with antigens. Although generally used to identify the constituents in protein complexes, the method can also be used to study the regulation of complex formation. As we have seen with anticancer agents129 and chaperones,130 a protein receptorcentered complex assembly can be studied through immune selection at various stages of complex formation. The fact that 40% or more of cellular proteins are probably in complexes makes this type of MALISA an important tool for studying an interactome, but refinement is needed. One of the problems is that members of low binding affinity can be eluted from a complex during affinity selection and evade detection.131 Others are that false positive binding is common. Reproducibility and prediction of binding partners have generally been poor.132 The good news is that new algorithms have recently been developed that predict true association partners with 80% sensitivity and a 0.3% false discovery rate.133 Type 2 MALISA. This type of assay is more common in immunohistochemistry than in traditional immunological assays. Type 2 MALISA immune capture is defined here as “selecting proteins in which a significant portion, or all of the epitope being targeted, is a PTM, generally using an mAb in immune complex formation”. This assay type is unique in that the protein features being targeted may not be tightly coupled to specific genes, multiple proteins can bear the same modification, and regulatory changes in multiple enzymes may be involved in the modification. The incidence with which protein modifications are involved in regulation and disease has greatly stimulated interest in identifying where they occur in nature and on what proteins. With phosphate ester-bearing proteins, for example, antibodies have been used to target either a tyrosine phosphate134−137 or, in the more protein-specific case, a phosphorylated amino acid surrounded by a unique amino acid sequence.138−140 Hundreds of phosphorylated proteins have 744



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expression product of a single gene by proteolysis and subsequent immune capture using either a single pAb or mAb”. The term “signature peptide” used in the above definition is a peptide unique to a single protein22 but common to all isoforms of the protein.155 It is also assumed that a signature peptide is a limit tryptic peptide, where the term limit means it is fully digested by trypsin. Confidence that the protein parent has been correctly identified is elevated by simultaneous determination of multiple signature peptides using multiple antibodies.22 For this reason, it is generally suggested that protein identification should be based on three signature peptides. Type 4 MALISA can be achieved by either MALDI-MS/MS or ESI-MS/MS. With MALDI-MS/MS peptides from the tryptic digest are generally analyzed without RPC fractionation between the immune capture and MS/MS steps.156 This has the limitation that the background from nonspecifically bound peptides will be high, and large-scale multiplexing will be more difficult. Type 4 assays by ESI-MS/MS, in contrast, are almost always accompanied by intermediate RPC fractionation before MS/ MS.28 Considering that RPC columns have peptide peak capacities of 100−450,157 this enormously increases the selectivity and multiplexing potential. Most type 4 MALISA applications have been in the SISCAPA mode, the great advantage being the achievement of high levels of quantification through multiple-reaction monitoring. In the assessment of cardiovascular injury with the troponin I (cTnI) and interleukin 33 (IL-33) biomarkers, determinations were linear from 1.5 to 5000 μg/mL.158 The dynamic range is exceptional in this case. Moreover, there was good correlation with commercial immunological assays in plasma across the clinically significant 1−10 mg/mL range. Another study focused on the development of a SISCAPA method for serum thyroglobulin as a potential tumor marker.159 With the use of polyclonal antibodies against three peptides, assays were developed with a limit of detection in the picomole range that showed good correlation with conventional immunoassays.

proteins that, according to databases, have one or more peptide sequences similar or identical to the targeted protein. Epitopes in this case will be of two types: (i) a continuous (linear) sequence of 6−10 amino acids derived from the primary structure of the protein or (ii) a discontinuous cluster of amino acids, the epitope being formed by single residues or short sequences at noncontiguous sites in the primary structure that are brought together during folding. Discontinuous epitopes arising from secondary and tertiary structures are dominant in most native proteins152 but are conformationally sensitive. Uhlen points out that in antibody-based proteomics, the proteins being captured and examined may be partially or fully unfolded; thus, the antibodies targeting continuous epitopes will be of the greatest value.150 A second advantage in using linear epitopes is that epitope-bearing peptides conjugated to immunogens can be used in immunization; there is no need for native proteins. The disadvantage is that linear epitopes can be hidden in the protein interior and unrecognizable, even though the protein is partially denatured. It will be important that peptides used in preparing antibodies be at the exterior of proteins. Concerns about the potential for cross-reactivity of 5−8 amino acid epitopes in immunological assays are well-founded. In silico analyses indicate that although unique linear epitopes can be found with the majority of human proteins, there is also widespread sequence similarity among other proteins.150 Even peptides differing by a single amino acid in otherwise identical sequences can have affinity for the same paratope. A comparison of the HIV-1 virus to the human proteome found at the pentapeptide level reveals that there are 14227 matches with 10312 human proteins. This drops to 50 matches at the heptapeptide level and 3 in the case of octapeptides.153 It is not surprising with this degree of similarity that proteins cross-react in immunological assays, and viruses or microorganisms can trigger autoimmunity and autoimmune diseases.154 Protein-targeting antibodies are best characterized by mixedmode MALISA, whereas peptide-directed antibodies, by necessity, can be evaluated only through the bottom-up mode. Top-down MALISA lacks the requisite separation and sequencing power to understand how or why cross-reactivity is occurring. The greatest problem with type 3 MALISA is in differentiating between nonspecific binding and cross-reactivity. It is generally assumed that the hundreds of nontargeted polypeptides often seen in MALISA spectra are from nonspecifically bound proteins or peptides and thus are of no consequence. But even if there is only a single cross-reacting epitope, it can have a dramatic impact on quantification by competing with the targeted antigen for the antibody site. Type 3 assays are likely to be mandated by federal regulatory agencies in future clinical assay validation and lot-to-lot quality control of antibodies. The potential of this method in the study of autoimmune diseases is yet to be determined. Type 4 Assays. MALISA based on antibody selection of peptides are classified here as type 4 assays. It has been noted that proteins and variants thereof possess a large number of structural features, some of which are shared by all the isoforms and others that are not. Moreover, the structural feature conveying biological activity may be shared by all the variants or belong to a single isoform. It is generally implied in the published work that the current peptide-based immunological assays are designed to target a unique feature in the primary structure of a protein that is common to all isoforms.22,28,127 Thus, the function of the immune capture component in type 4 MALISA is defined as “selecting a signature peptide derived from the

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CONCLUSIONS This review began with a series of questions many scientists have today regarding the potential of MS as a tool for routine analysis of “known” proteins in blood and other complex samples. What emerged is the revelation that sample preparation protocols have a major impact on what can be seen with mass-spectral detection. Many proteins are not really known, in view of the fact that they exist in a variety of isoforms of poorly defined structure and function. Moreover, the structural feature responsible for their biological activity may not be known and may be different than the features being quantified in a mixture of isoforms. Another critical issue is that isoforms may not be of equal biological activity. Isoform differentiation will be a critical component of immunological assays in the future. Finally, it was shown that much of the discrimination between isoforms has to do with how the sample-preparation steps are linked to MS analysis. A major question in the future of immunological assays is not so much the potential of MS as how samples can be prepared to fully exploit MS. We conclude that when coupled to antibody selection of an analyte in a carefully selected set of steps, an integrated MALISA system is vastly superior to any other known technology in targeting molecular features of a protein. This derives from the ability of an antibody to capture polypeptides bearing a single or a few unique structural features that might simplify and enrich an analyte ≥1000-fold. When this greatly simplified sample is then 745



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(2) Liu, J.; Hewel, J. A.; Fong, V.; Chan-Shen-Yue, M.; Emili, A. Clin. Proteomics 2009, 5 (1), 3−14. (3) Lin, Y.; Dynan, W. S.; Lee, J. R.; Zhu, Z.-H.; Schade, R. R. Dig. Dis. Sci. 2009, 54 (3), 431−457. (4) Tian, T.; Hao, J.; Xu, A.; Hao, J.; Luo, C.; Liu, C.; Huang, L.; Xiao, X.; He, D. Cancer Sci. 2007, 98 (8), 1265−1274. (5) Jones, A. W.; Cooper, H. J. Analyst 2011, 136 (17), 3419−3429. (6) Ytterberg, J. A.; Dunsmore, J.; Lomeli, S. H.; Thevis, M.; Xie, Y.; Ogorzalek Loo, R. R.; Loo, J. A. InElectrospray and MALDI Mass Spectrometry: Fundamentals, Instrumentation, Practicalities, and Biological Applications, 2nd ed.; Cole, R. B., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2010; pp 685−721. (7) Peaston, R. T.; Weinkove, C. Ann. Clin. Biochem. 2004, 41 (1), 17− 38. (8) Pathak, S. S.; Tibbe, G. M.; Savelkoul, H. F. J. Immunol. Methods Man. 1997, 2, 1077−1094. (9) Kummer, J. A.; Strik, M. C. M.; Bladergroen, B. A.; Hack, C. E. Methods 2004, 32 (2), 141−149. (10) Ackermann, B. L.; Berna, M. J. Expert Rev. Proteomics 2007, 4 (2), 175−186. (11) Levin, R. E. Food Biotechnol. 2009, 23 (3), 214−228. (12) Janis, L. J.; Regnier, F. E. J. Chromatogr. 1988, 444, 1−11. (13) Desport, M.; Lewis, J. Curr. HIV Res. 2010, 8 (1), 53−65. (14) Roddam, A. W.; Duffy, M. J.; Hamdy, F. C.; Ward, A. M.; Patnick, J.; Price, C. P.; Rimmer, J.; Sturgeon, C.; White, P.; Allen, N. E. Eur. Urol. 2005, 48, 386−399. (15) Nedelkov, D. Expert Rev. Proteomics 2006, 3 (6), 631−640. (16) Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67 (24), 4581− 4585. (17) Yan, H.; Ahmad-Tajudin, A.; Bengtsson, M.; Xiao, S.; Laurell, T.; Ekstroem, S. Anal. Chem. 2011, 83 (12), 4942−4948. (18) Merchant, M.; Weinberger, S. R. Electrophoresis 2000, 21 (6), 1164−1177. (19) Kuwata, H.; Yip, T.-T.; Yip, C. L.; Tomita, M.; Hutchens, T. W. Adv. Exp. Med. Biol. 1998, 443, 23−32. (20) Bailes, J.; Soloviev, M. Peptidomics 2008, 191−231. (21) Nicol, G. R.; Han, M.; Kim, J.; Birse, C. E.; Brand, E.; Nguyen, A.; Mesri, M.; FitzHugh, W.; Kaminker, P.; Moore, P. A.; Ruben, S. M.; He, T. Circ. Res. 2009, 104 (1), 32−40. (22) Anderson, N. L.; Anderson, N. G.; Haines, L. R.; Hardie, D. B.; Olafson, R. W.; Pearson, T. W. J. Proteome Res. 2004, 3 (2), 235−244. (23) Nicol, G. R.; Han, M.; Kim, J.; Birse, C. E.; Brand, E.; Nguyen, A.; Mesri, M.; FitzHugh, W.; Kaminker, P.; Moore, P. A.; Ruben, S. M.; He, T. Circ. Res. 2010, 10 (20), 3566−3576. (24) Han, X.; Aslanian, A.; Yates, J. R., III Curr. Opin. Chem. Biol. 2008, 12 (5), 483−490. (25) Tanner, S. D.; Bandura, D. R.; Ornatsky, O.; Baranov, V. I.; Nitz, M.; Winnik, M. A. Pure Appl. Chem. 2008, 80 (12), 2627−2641. (26) Alexander, J. E.; Hunt, D. F.; Lee, M. K.; Shabanowitz, J.; Michel, H.; Berlin, S. C.; MacDonald, T. L.; Sundberg, R. J.; Rebhun, L. I.; Frankfurter, A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88 (11), 4685−4689. (27) Papac, D. I.; Hayes, J.; Tomer, K. B. Anal. Chem. 1994, 66, 2609− 2613. (28) Krone, J. R.; Nelson, R. W.; Dogruel, D.; Williams, P.; Granzow, R. BIA J 1996, 3 (1), 16−17. (29) Hutchens, T. W.; Yip, T. T. Rapid Commun. Mass Spectrom. 1993, 7, 576−580. (30) Niederkofler, E. E.; Kiernan, U. A.; O’Rear, J.; Menon, S.; Syed, S. Journal American Heart Association 2008, 1, 258−264. (31) Wolters, D. A.; Washburn, M. P.; Yates, J. R., III Anal. Chem. 2001, 73 (23), 5683−5690. (32) Whiteaker, J. R.; Zhao, L.; Abbatiello, S. E.; Burgess, M.; Kuhn, E.; Lin, C. W.; Pope, M. E.; Razavi, M.; Anderson, N. L.; Pearson, T. W.; Carr, S. A.; Paulovich, A. G. Mol. Cell. Proteomics 2011, 10 (4), 5645/1− 5645/10. (33) Tanaka, K.; Kaki, H.; Ido, Y. Rapid Commun. Mass Spectrom. 1988, 2, 151−153. (34) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60 (20), 2299−2301.

introduced into either a MALDI-MS/MS or a LC-MS/MS instrument, there will be sufficient concentration and separation space to discriminate among other structural features of the analyte. There is synergy in the mode of linkage. Moreover, the MS instrument is capable of doing this with multiple analytes simultaneously within seconds, when samples are properly prepared. We further conclude that analytical methods, such as ELISA, that fail to discriminate among isoforms, without validation that all the components being assessed play a role in a biological phenomenon, are at high risk of providing erroneous results. Finally, we conclude that the Achilles' heel of MALISA is its sensitivity. MALISA methods are at least 3−4 orders of magnitude less sensitive than the most sensitive ELISA methods. MS-based immunological-assay methods will have difficulty displacing traditional immunological assays until they are of equivalent sensitivity.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors share equal contribution.

Notes

The authors declare no competing financial interest. Biographies Dr. Ashraf G. Madian received his B.Sc. (2001) in Pharmaceutical Sciences from Ain Shams University, Cairo, Egypt, and M.Sc. in Pharmaceutical and Chemical Sciences from the University of the Pacific, Stockton, CA. He received his Ph.D. in 2010 in Analytical Chemistry, under the supervision of Dr. Fred E. Regnier at Purdue University. His research at Purdue focused on the development of technologies to diagnose diseases and evaluate therapeutic agents based on the detection of oxidized polypeptides. He is currently a Fellow in the Pritzker School of Medicine at the University of Chicago. Dr. Nishi S. Rochelle is currently a postdoctoral researcher in the Department of Medicinal Chemistry and Molecular Pharmacology at Purdue University. She graduated with her B.S. in Chemistry from Xavier University of Louisiana in 2005 and received her Ph.D. in Analytical Chemistry from Purdue University in 2012, under the supervision of Dr. Fred E. Regnier. Her research interests include method development for detection and quantification of posttranslationally modified proteins important for prognostic and diagnostic applications in cancer. Dr. Fred E. Regnier is a Distinguished Professor of Chemistry at Purdue University. He is generally regarded as one of the world authorities on proteomics. Additionally, he is an accomplished entrepreneur, cofounding several companies such as Bioseparations, PerSeptive Biosystems, Beyond Genomics, and Perfinity Biosystems. He also serves as a Member of the Scientific Advisory Council at Tienta Sciences, Inc. He has published more than 300 journal articles and 70 book chapters and reviews, has more than 40 patents, has edited two books, and has won numerous national and international awards and distinctions for his research in analytical chemistry and biochemistry. Dr. Regnier received his B.S. from the Nebraska State University and holds a Ph.D. from the University of Oklahoma. He did his postdoctoral training at the University of Chicago and Harvard University.

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