Advances and Utility of the Human Plasma Proteome.

The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises 4395 canonical and 1482 additional nonredundant human proteins detected in 240 MS-based experiments. In addition, we report the new Human Extracellular Vesicle PeptideAtlas 2021-06, which comprises five studies and 2757 canonical proteins detected in extracellular vesicles circulating in blood, of which 74% (2047) are in common with the plasma PeptideAtlas. Our overview summarizes the recent advances, impactful applications, and ongoing challenges for translating plasma proteomics into utility for precision medicine.

Mass Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data.

The proteomic analysis of human blood and blood-derived products (e.g., plasma) offers an attractive avenue to translate research progress from the laboratory into the clinic. However, due to its unique protein composition, performing proteomics assays with plasma is challenging. Plasma proteomics has regained interest due to recent technological advances, but challenges imposed by both complications inherent to studying human biology (e.g., interindividual variability) and analysis of biospecimens (e.g., sample variability), as well as technological limitations remain. As part of the Human Proteome Project (HPP), the Human Plasma Proteome Project (HPPP) brings together key aspects of the plasma proteomics pipeline. Here, we provide considerations and recommendations concerning study design, plasma collection, quality metrics, plasma processing workflows, mass spectrometry (MS) data acquisition, data processing, and bioinformatic analysis. With exciting opportunities in studying human health and disease though this plasma proteomics pipeline, a more informed analysis of human plasma will accelerate interest while enhancing possibilities for the incorporation of proteomics-scaled assays into clinical practice.

High-quality MS/MS spectrum prediction for data-dependent and data-independent acquisition data analysis.

Peptide fragmentation spectra are routinely predicted in the interpretation of mass-spectrometry-based proteomics data. However, the generation of fragment ions has not been understood well enough for scientists to estimate fragment ion intensities accurately. Here, we demonstrate that machine learning can predict peptide fragmentation patterns in mass spectrometers with accuracy within the uncertainty of measurement. Moreover, analysis of our models reveals that peptide fragmentation depends on long-range interactions within a peptide sequence. We illustrate the utility of our models by applying them to the analysis of both data-dependent and data-independent acquisition datasets. In the former case, we observe a q-value-dependent increase in the total number of peptide identifications. In the latter case, we confirm that the use of predicted tandem mass spectrometry spectra is nearly equivalent to the use of spectra from experimental libraries.

Chemical Glycoproteomics.

Chemical tools have accelerated progress in glycoscience, reducing experimental barriers to studying protein glycosylation, the most widespread and complex form of posttranslational modification. For example, chemical glycoproteomics technologies have enabled the identification of specific glycosylation sites and glycan structures that modulate protein function in a number of biological processes. This field is now entering a stage of logarithmic growth, during which chemical innovations combined with mass spectrometry advances could make it possible to fully characterize the human glycoproteome. In this review, we describe the important role that chemical glycoproteomics methods are playing in such efforts. We summarize developments in four key areas: enrichment of glycoproteins and glycopeptides from complex mixtures, emphasizing methods that exploit unique chemical properties of glycans or introduce unnatural functional groups through metabolic labeling and chemoenzymatic tagging; identification of sites of protein glycosylation; targeted glycoproteomics; and functional glycoproteomics, with a focus on probing interactions between glycoproteins and glycan-binding proteins. Our goal with this survey is to provide a foundation on which continued technological advancements can be made to promote further explorations of protein glycosylation.

Direct detection of nitrotyrosine-containing proteins using an aniline-based oxidative coupling strategy.

A convenient two-step method is described for the detection of nitrotyrosine-containing proteins. First, nitrotyrosines are reduced to aminophenols using sodium dithionite. Following this, an oxidative coupling reaction is used to attach anilines bearing fluorescence reporters or affinity probes. Features of this approach include fast reaction times, pmol-level sensitivity, and excellent chemoselectivity.

Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis.

Protein glycosylation is a heterogeneous post-translational modification (PTM) that plays an essential role in biological regulation. However, the diversity found in glycoproteins has undermined efforts to describe the intact glycoproteome via mass spectrometry (MS). We present IsoTaG, a mass-independent chemical glycoproteomics platform for characterization of intact, metabolically labeled glycopeptides at the whole-proteome scale. In IsoTaG, metabolic labeling of the glycoproteome is combined with (i) chemical enrichment and isotopic recoding of glycopeptides to select peptides for targeted glycoproteomics using directed MS and (ii) mass-independent assignment of intact glycopeptides. We structurally assigned 32 N-glycopeptides and over 500 intact and fully elaborated O-glycopeptides from 250 proteins across three human cancer cell lines and also discovered unexpected peptide sequence polymorphisms (pSPs). The IsoTaG platform is broadly applicable to the discovery of PTM sites that are amenable to chemical labeling, as well as previously unknown protein isoforms including pSPs.

A chemical glycoproteomics platform reveals O-GlcNAcylation of mitochondrial voltage-dependent anion channel 2.

Protein modification by O-linked ß-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling modality, but identifying signal-specific O-GlcNAcylation events remains a significant experimental challenge. Here, we describe a method for visualizing and analyzing organelle- and stimulus-specific O-GlcNAcylated proteins and use it to identify the mitochondrial voltage-dependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2(-/-) cells resist the mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation, demonstrating a functional connection between O-GlcNAc signaling and mitochondrial physiology through VDAC2. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts.

HyperCEST detection of a 129Xe-based contrast agent composed of cryptophane-A molecular cages on a bacteriophage scaffold.

A hyperpolarized 129Xe contrast agent composed of many cryptophane-A molecular cages assembled on an M13 bacteriophage has been demonstrated. Saturation of xenon bound in the large number of cryptophane cages is transferred to the pool of aqueous-solvated xenon via chemical exchange, resulting in efficient generation of hyperCEST contrast. No significant loss of contrast per cryptophane cage was observed for the multivalent phage when compared with unscaffolded cryptophane. Detection of this phage-based hyperCEST agent is reported at concentrations as low as 230 fM, representing the current lower limit for NMR/MRI-based contrast agents.

Mapping yeast N-glycosites with isotopically recoded glycans.

Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome.

Isotopic signature transfer and mass pattern prediction (IsoStamp): an enabling technique for chemically-directed proteomics.

Directed proteomics applies mass spectrometry analysis to a subset of information-rich proteins. Here we describe a method for targeting select proteins by chemical modification with a tag that imparts a distinct isotopic signature detectable in a full-scan mass spectrum. Termed isotopic signature transfer and mass pattern prediction (IsoStamp), the technique exploits the perturbing effects of a dibrominated chemical tag on a peptide's mass envelope, which can be detected with high sensitivity and fidelity using a computational method. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. To identify a tagged-peptide's sequence, we performed an inclusion list-driven shotgun proteomics experiment where peptides bearing a recoded mass envelope were targeted for fragmentation, allowing for direct site mapping. Using this approach, femtomole quantities of several targeted peptides were identified in total mammalian cell lysate, while traditional data-dependent methods were unable to identify as many peptides. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). IsoStamp has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for posttranslational modification.

A chemical method for labeling lysine methyltransferase substrates.

Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

MRI thermometry based on encapsulated hyperpolarized xenon.

A new approach to MRI thermometry using encapsulated hyperpolarized xenon is demonstrated. The method is based on the temperature dependent chemical shift of hyperpolarized xenon in a cryptophane-A cage. This shift is linear with a slope of 0.29 ppm °C(-1) which is perceptibly higher than the shift of the proton resonance frequency of water (ca. 0.01 ppm °C(-1)) that is currently used for MRI thermometry. Using spectroscopic imaging techniques, we collected temperature maps of a phantom sample that could discriminate by direct NMR detection between temperature differences of 0.1 °C at a sensor concentration of 150 µM. Alternatively, the xenon-in-cage chemical shift was determined by indirect detection using saturation transfer techniques (Hyper-CEST) that allow detection of nanomolar agent concentrations. Thermometry based on hyperpolarized xenon sensors improves the accuracy of currently available MRI thermometry methods, potentially giving rise to biomedical applications of biosensors functionalized for binding to specific target molecules.

A xenon-based molecular sensor assembled on an MS2 viral capsid scaffold.

In MRI, anatomical structures are most often differentiated by variations in their bulk magnetic properties. Alternatively, exogenous contrast agents can be attached to chemical moieties that confer affinity to molecular targets; the distribution of such contrast agents can be imaged by magnetic resonance. Xenon-based molecular sensors are molecular imaging agents that rely on the reversible exchange of hyperpolarized xenon between the bulk and a specifically targeted host-guest complex. We have incorporated approximately 125 xenon sensor molecules in the interior of an MS2 viral capsid, conferring multivalency and other properties of the viral capsid to the sensor molecule. The resulting signal amplification facilitates the detection of sensor at 0.7 pM, the lowest to date for any molecular imaging agent used in magnetic resonance. This amplification promises the detection of chemical targets at much lower concentrations than would be possible without the capsid scaffold.

Probing the binding site of the A1 adenosine receptor reengineered for orthogonal recognition by tailored nucleosides.

His272 (7.43) in the seventh transmembrane domain (TM7) of the human A3 adenosine receptor (AR) interacts with the 3' position of nucleosides, based on selective affinity enhancement at a H272E mutant A3 AR (neoceptor) of 3'-ureido, but not 3'-OH, adenosine analogues. Here, mutation of the analogous H278 of the human A1 AR to Ala, Asp, Glu, or Leu enhanced the affinity of novel 2'- and 3'-ureido adenosine analogues, such as 10 (N6-cyclopentyl-3'-ureido-3'-deoxyadenosine), by >100-fold, while decreasing the affinity or potency of adenosine and other 3'-OH adenosine analogues. His278 mutant receptors produced a similar enhancement regardless of the charge character of the substituted residue, implicating steric rather than electrostatic factors in the gain of function, a hypothesis supported by rhodopsin-based molecular modeling. It was also demonstrated that this interaction was orientationally specific; i.e., mutations at the neighboring Thr277 did not enhance the affinity for a series of 2'- and 3'-ureido nucleosides. Additionally, H-bonding groups placed on substituents at the N6 or 5' position demonstrated no enhancement in the mutant receptors. These reengineered human A1 ARs revealed orthogonality similar to that of the A3 but not the A2A AR, in which mutation of the corresponding residue, His278, to Asp did not enhance nucleoside affinity. Functionally, the H278D A1 AR was detectable only in a measure of membrane potential and not in calcium mobilization. This neoceptor approach should be useful for the validation of molecular modeling and the dissection of promiscuous GPCR signaling.

Discovery of a new nucleoside template for human A3 adenosine receptor ligands: D-4'-thioadenosine derivatives without 4'-hydroxymethyl group as highly potent and selective antagonists.

Truncated D-4'-thioadenosine derivatives lacking the 4'-hydroxymethylene moiety were synthesized starting from D-mannose, using cyclization to the 4-thiosugar and one-step conversion of the diol to the acetate as key steps. At the human A3 adenosine receptor (AR), N6-substituted purine analogues bound potently and selectively and acted as antagonists in a cyclic AMP functional assay. An N6-(3-chlorobenzyl)purine analogue 9b displayed a Ki value of 1.66 nM at the human A3 AR. Thus, truncated D-4'-thioadenosine is an excellent template for the design of novel A3 AR antagonists to act at both human and murine species.

Structure-activity relationships of 2,N(6),5'-substituted adenosine derivatives with potent activity at the A2B adenosine receptor.

2, N6, and 5'-substituted adenosine derivatives were synthesized via alkylation of 2-oxypurine nucleosides leading to 2-arylalkylether derivatives. 2-(3-(Indolyl)ethyloxy)adenosine 17 was examined in both binding and cAMP assays and found to be a potent agonist of the human A2BAR. Simplification, altered connectivity, and mimicking of the indole ring of 17 failed to maintain A2BAR potency. Introduction of N6-ethyl or N6-guanidino substitution, shown to favor A2BAR potency, failed to enhance potency in the 2-(3-(indolyl)ethyloxy)adenosine series. Indole 5' '- or 6' '-halo substitution was favored at the A2BAR, but a 5'-N-ethylcarboxyamide did not further enhance potency. 2-(3' '-(6' '-Bromoindolyl)ethyloxy)adenosine 28 displayed an A2BAR EC50 value of 128 nM, that is, more potent than the parent 17 (299 nM) and similar to 5'-N-ethylcarboxamidoadenosine (140 nM). Compound 28 was a full agonist at A2B and A2AARs and a low efficacy partial agonist at A1 and A3ARs. Thus, we have identified and optimized 2-(2-arylethyl)oxo moieties in AR agonists that enhance A2BAR potency and selectivity.

2-triazole-substituted adenosines: a new class of selective A3 adenosine receptor agonists, partial agonists, and antagonists.

"Click chemistry" was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1-14). Binding affinity at the human A(1), A(2A), and A(3)ARs (adenosine receptors) and relative efficacy at the A(3)AR were determined. Some triazol-1-yl analogues showed A(3)AR affinity in the low nanomolar range, a high ratio of A(3)/A(2A) selectivity, and a moderate-to-high A(3)/A(1) ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A(3)AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A(3)AR efficacy. Thus, several 5'-OH derivatives appeared to be selective A(3)AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A(1)AR and displaying a characteristic docking mode in an A(3)AR model. The corresponding 5'-ethyluronamide analogues generally showed increased A(3)AR affinity and behaved as full agonists, i.e., 17, with 910-fold A(3)/A(1) selectivity. Thus, N(6)-substituted 2-(1,2,3-triazolyl)adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A(3)AR antagonists, partial agonists, and agonists.

NUCLEOSIDE PRODRUGS OF A3 ADENOSINE RECEPTOR AGONISTS AND ANTAGONISTS.

9-(ß-D-Ribosfuranosyluronamide)adenine derivatives that are selective agonists and antagonists of the A3 adenosine receptor (AR) have been derivatized as prodrugs for in vivo delivery. The free hydroxy groups at the 2' and 3' positions of the agonists 2-chloro-N 6-(3-iodobenzyl)-9-(N-methyl-(ß-D-ribosfuranosyluronamide)adenine 2b, the corresponding 4'-thio nucleoside 2c, and antagonists 4a and 4b (5'-N,N-dimethylamides related to 2b and 2c, respectively) were derivatized through simple acylation reactions. The prodrug derivatives were tested in radioligand binding assays at ARs and in a functional assay of adenylate cyclase at the A3AR and found to be considerably less active than the parent drugs. The hydrolysis of nucleoside 2',3'-diesters to regenerate the parent compound in the presence of human blood was demonstrated. 2',3'-Dipropionate esters of 2b and 4a were readily cleaved in a two-step reaction to regenerate the parent drug, on a time scale of two hours. The cleavage of a 2',3'-dihexanoate ester occurred at a slower rate. This indicates that the prodrugs are suitable as masked forms of the biologically active A3AR agonists and antagonists for future evaluation in vivo.