Intact Protein Mass Spectrometry Research Articles

Deconvolving Native and Intact Protein Mass Spectra with UniDec.

Intact protein, top-down, and native mass spectrometry (MS) generally requires the deconvolution of electrospray ionization (ESI) mass spectra to assign the mass of components from their charge state distribution. For small, well-resolved proteins, the charge can usually be assigned based on the isotope distribution. However, it can be challenging to determine charge states with larger proteins that lack isotopic resolution, in complex mass spectra with overlapping charge states, and in native spectra that show adduction. To overcome these challenges, UniDec uses Bayesian deconvolution to assign charge states and to create a zero-charge mass distribution. UniDec is fast, user-friendly, and includes a range of advanced tools to assist in intact protein, top-down, and native MS data analysis. This chapter provides a step-by-step protocol and an in-depth explanation of the UniDec algorithm, and highlights the parameters that affect the deconvolution. It also covers advanced data analysis tools, such as macromolecular mass defect analysis and tools for assigning potential PTMs and bound ligands. Overall, this chapter provides users with a deeper understanding of UniDec, which will enhance the quality of deconvolutions and allow for more intricate MS experiments.

The antibody-binding Fc gamma receptor IIIa / CD16a is N-glycosylated with high occupancy at all five sites

The antibody-binding Fc γ receptors (FcγRs) trigger life-saving immune responses and many therapeutic monoclonal antibodies require FcγR engagement for full effect. One proven strategy to improve the efficacy of antibody therapies is to increase receptor binding affinity, in particular binding to FcγRIIIa/CD16a. Currently, affinities are measured using recombinantly-expressed soluble extracellular FcγR domains and CD16a-mediated antibody-dependent immune responses are characterized using cultured cells. It is notable that CD16a is highly processed with multiple N-glycosylation sites, and preventing individual N-glycan modifications affects affinity. Furthermore, multiple groups have demonstrated that CD16a N-glycan composition is variable and composition impacts antibody binding affinity. The level of N-glycosylation at each site is not known though computational prediction indicates low to moderate potential at each site based on primary sequence (40–70%). Here we quantify occupancy of the extracellular domains using complementary mass spectrometry-based methods. All five sites of the tighter-binding CD16a V158 allotype showed 65–100% N-glycan occupancy in proteomics-based experiments. These observations were confirmed using intact protein mass spectrometry that demonstrated the predominant species corresponded to CD16a V158 with five N-glycans, with a smaller contribution from CD16a with four N-glycans. Occupancy was likewise high for the membrane-bound receptor at all detected N-glycosylation sites using CD16a purified from cultured human natural killer cells. Occupancy of the N162 site, critical for antibody binding, appeared independent of N169 occupancy based on analysis of the T171A mutant protein. The weaker-binding CD16a F158 allotype showed higher occupancy of >93% at each site.

Mass spectrometry approaches for the selection of primary calibrators in the development of reference method

AbstractBackgroundNeurofilament‐light chain (NF‐L) is a known early marker for neurodegeneration. This marker which is essential for the axonal structure, is quantifiable in biological fluids after neuronal damage but is not specific to a given pathology. Currently, the methods for detecting/quantifying this biomarker are mainly based on immunoassays. While the performance of immunoassay‐based assays is good in term of sensitivity and precision, mass spectrometry (MS) approaches are required to achieve standardisation. Those aiding to improved characterisation/quantification of the calibrator and potential for developing a reference measurement procedure traceable to the International System of Units. Furthermore MS has the potential of multiplexing NF‐L with other specific biomarkers for dementia. The characterization and quantification of a number of primary calibrators and their suitability for harmonization of immunoassay based results and development of a reference measurement procedure is here assessed.MethodMicrowaver hydrolysis and isotope dilution MS was used for quantification of NF‐L from different manufactures. An Orbitrap mass spectrometer (Q‐Exactive, Thermofischer) was used to provide high resolution intact protein analysis information. A QQQ mass spectrometer (Xevo TQ‐XS, Waters) was utilized to assess the potential of developing a reference measurement procedure for NF‐L.ResultThe quantitative analysis of the three NF‐L by amino acid analysis (AAA) showed for all samples discrepancy between the nominal concentrations and the experimental results with a general overestimation for the nominal concentration due to water and salt contents. Separation, identification and quantification of impurities present in the preparations was attempted to obtain a total protein content from the AAA and the NF‐L content from the AAA results corrected for the impurities. The sequence of NF‐L was verified by high resolution intact protein MS and tryptic digestion. A QQQ method was developed to monitor the tryptic digest peptides and assess their suitability to be used for the development of a reference measurement procedure. This was based on specificity, limits of detection and limits of quantification.ConclusionThese studies allow us to characterize three NF‐L proteins from different suppliers and to perform a first selection of the most relevant ones as primary calibrators for the development of a reference method.

Poly(acrylamide-co-N,N'-methylenebisacrylamide) Monoliths for High-Peak-Capacity Hydrophilic-Interaction Chromatography-High-Resolution Mass Spectrometry of Intact Proteins at Low Trifluoroacetic Acid Content.

In this study, we optimized a polymerization mixture to synthesize poly(acrylamide-co-N,N′-methylenebisacrylamide) monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of cross-linker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy (SEM). Further characterization of the monolithic structure revealed a permeable (Kf between 2.5 × 10–15 and 1.40 × 10–13 m2) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 μm core–shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA), the peak capacities of the two columns were similar (30 and 34 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained similar separation performance and selectivity (peak capacity 23), whereas the packed column showed greatly reduced performance (peak capacity 12), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA, the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.

Acetylation of the Catalytic Lysine Inhibits Kinase Activity in PI3Kδ

Covalent inhibition is a powerful strategy to develop potent and selective small molecule kinase inhibitors. Targeting the conserved catalytic lysine is an attractive method for selective kinase inactivation. We have developed novel, selective inhibitors of phosphoinositide 3-kinase δ (PI3Kδ) which acylate the catalytic lysine, Lys779, using activated esters as the reactive electrophiles. The acylating agents were prepared by adding the activated ester motif to a known selective dihydroisobenzofuran PI3Kδ inhibitor. Three esters were designed, including an acetate ester which was the smallest lysine modification evaluated in this work. Covalent binding to the enzyme was characterized by intact protein mass spectrometry of the PI3Kδ-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδ inhibition was a direct result of covalent lysine acylation. These results indicate that a simple chemical modification such as lysine acetylation is sufficient to inhibit kinase activity. The selectivity of the compounds was evaluated against lipid kinases in cell lysates using a chemoproteomic binding assay. Due to the conserved nature of the catalytic lysine across the kinome, we believe the covalent inhibition strategy presented here could be applicable to a broad range of clinically relevant targets.

Internal Fragments Generated from Different Top-Down Mass Spectrometry Fragmentation Methods Extend Protein Sequence Coverage.

Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N- or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photon-based fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome c, myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15-20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.

Semiprocessive Hyperglycosylation of Adhesin by Bacterial Protein N-Glycosyltransferases.

Processivity is an important feature of enzyme families such as DNA polymerases, polysaccharide synthases, and protein kinases, to ensure high fidelity in biopolymer synthesis and modification. Here, we reveal processive character in the family of cytoplasmic protein N-glycosyltransferases (NGTs). Through various activity assays, intact protein mass spectrometry, and proteomics analysis, we established that NGTs from nontypeable Haemophilus influenzae and Actinobacillus pleuropneumoniae modify an adhesin protein fragment in a semiprocessive manner. Molecular modeling studies suggest that the processivity arises from the shallow substrate binding groove in NGT, which promotes the sliding of the adhesin over the surface to allow further glycosylations without temporary dissociation. We hypothesize that the processive character of these bacterial protein glycosyltransferases is the mechanism to ensure multisite glycosylation of adhesins in vivo, thereby creating the densely glycosylated proteins necessary for bacterial self-aggregation and adherence to human cells, as a first step toward infection.

Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5.

Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5:MEP50 uses a rapid equilibrium random order mechanism with EAP and EBQ dead-end complexes. We also provide evidence of ternary complex formation in solution using hydrogen/deuterium exchange mass spectrometry. Isotope exchange and intact protein mass spectrometry further rule out ping-pong as a potential enzyme mechanism, and finally, we show that PRMT5 exhibits a pre-steady state burst that corresponds to an initial slow turnover with all four active sites of the hetero-octamer being catalytically active.

Dissecting Total Plasma and Protein-Specific Glycosylation Profiles in Congenital Disorders of Glycosylation.

Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.

Intact Protein Mass Spectrometry for Therapeutic Protein Quantitation, Pharmacokinetics, and Biotransformation in Preclinical and Clinical Studies: An Industry Perspective.

Recent advancements in immunocapture methods and mass spectrometer technology have enabled intact protein mass spectrometry to be applied for the characterization of antibodies and other large biotherapeutics from in-life studies. Protein molecules have not been traditionally studied by intact mass or screened for catabolites in the same manner as small molecules, but the landscape has changed. Researchers have presented methods that can be applied to the drug discovery and development stages, and others are exploring the possibilities of the new approaches. However, a wide variety of options for assay development exists without clear recommendation on best practice, and data processing workflows may have limitations depending on the vendor. In this perspective, we share experiences and recommendations for current and future application of mass spectrometry for biotherapeutic molecule monitoring from preclinical and clinical studies.

Investigating protein thiol chemistry associated with dehydroascorbate, homocysteine and glutathione using mass spectrometry.

Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.

LESA Cyclic Ion Mobility Mass Spectrometry of Intact Proteins from Thin Tissue Sections.

Liquid extraction surface analysis (LESA) is an ambient surface sampling technique that allows the analysis of intact proteins directly from tissue samples via mass spectrometry. Integration of ion mobility separation to LESA mass spectrometry workflows has shown significant improvements in the signal-to-noise ratios of the resulting protein mass spectra and hence the number of proteins detected. Here, we report the use of a quadrupole–cyclic ion mobility–time-of-flight mass spectrometer (Q-cIM-ToF) for the analysis of proteins from mouse brain and rat kidney tissues sampled via LESA. Among other features, the instrument allows multiple pass cyclic ion mobility separation, with concomitant increase in resolving power. Single-pass experiments enabled the detection of 30 proteins from mouse brain tissue, rising to 44 when quadrupole isolation was employed. In the absence of ion mobility separation, 21 proteins were detected in rat kidney tissue including the abundant α- and β-globin chains from hemoglobin. Single-pass cyclic ion mobility mass spectrometry enabled the detection of 60 additional proteins. Multipass experiments of a narrow m/z range (m/z 870–920) resulted in the detection of 24 proteins (one pass), 37 proteins (two passes) and 54 proteins (three passes), thus demonstrating the benefits of improved mobility resolving power.

A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of hOAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1R,3S,4S)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (1), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying 8 and 9 as novel selective hOAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated 8 and 9 as covalent inhibitors of hOAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel addition-aromatization inactivation mechanism for 9 contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.

Voltage Rollercoaster Filtering of Low-Mass Contaminants During Native Protein Analysis

Intact protein mass spectrometry (MS) via electrospray-based methods is often degraded by low-mass contaminants, which can suppress the spectral quality of the analyte of interest via space-charge effects. Consequently, selective removal of contaminants by their mobilities would benefit native MS if achieved without additional hardware and before the mass analyzer regions used for selection, analyte readout, or tandem MS. Here, we use the high-pressure multipole within the source of an Orbitrap Tribrid as the foundation for a coarse ion filter. Using this method, we show complete filtration of 2 mM polyethylene glycol (PEG-1000) during native MS of SILu mAb antibody present at a 200× lower concentration. We also show the generality of the process by rescuing 10 μM tetrameric pyruvate kinase from 2 mM PEG-1000, asserting this voltage rollercoaster filtering (VRF) method for use in native MS as an efficient alternative to conventional purification methods.

Quick and facile preparation of histone proteins from the green microalga Chlamydomonas reinhardtii and other photosynthetic organisms.

The development of universal, broadly applicable methods for histone extraction from animal cells and tissues has unlocked the ability to compare these epigenetic-influencing proteins across tissue types, healthy and diseased states, and cancerous versus normal cells. However, for plants and green algae, a quick and easily implemented histone extraction method has yet to be developed. Here, we report an optimized method that provides a unified approach to extract histones for the green microalgal species Chlamydomonas reinhardtii and Scenedesmus dimorphus as well as for maize (corn) leaf tissue. Histone extraction methods include treatment with high salt concentrations and acidification. Preparations of nuclei can be made in ∼3.5 h and histones extracted in ∼3.5 h either immediately or nuclei may be frozen and histone proteins can be later extracted without a change in histone PTM patterns. To examine the efficiency of the new methods provided, we performed both qualitative and quantitative analysis of salt and acid-extracted whole histone proteins (SAEWH) via SDS-PAGE gel electrophoresis and intact protein mass spectrometry. SDS-PAGE analysis indicated that histone yields decrease when using walled Chlamydomonas strains relative to cell-wall-less mutants. Using top-down mass spectrometry (TDMS) for intact protein analysis, we confirmed the presence of H4K79me1 in multiple algal species; however, this unique modification was not identified in corn leaf tissue and has not been reported elsewhere. TDMS measurements of SAEWH extracts also revealed that oxidation which occurs during the histone extraction process does not increase with exposure of harvested algal cells, their nuclei and the extracted histone samples to light.

Intact Protein Mass Spectrometry of Cell Culture Harvest Guides Cell Line Development for Trispecific Antibodies.

IgG-like multispecific antibodies with asymmetric constructs have become widely used formats for therapeutic applications in recent years. Correct assembly of the subunits in this class of therapeutics is a critical quality attribute (CQA) with direct impact on biological activity. Therefore, early drug development efforts such as clone selection during cell line development must be guided by information on potential chain mispairing to enable timely decision making and risk mitigation. Here we describe a high-throughput analytical platform based on denaturing size-exclusion ultraperformance liquid chromatography (UPLC) coupled with intact protein mass spectrometry for profiling of mispairing and other product-related impurities, including half antibodies. This method can be performed directly on the clarified cell culture harvest fluid without the need for Protein A purification or other sample preparations and provides unbiased information on the product quality of the clones and the effect of growth conditions in a fast and cost-effective manner. Screening large numbers of clones expressing different trispecific antibody (tsAb) constructs revealed that although chain mispairing primarily depends on the antibody sequence and structure, it is also a characteristic of the clone. In addition, different growth conditions may affect the type and distribution of half antibodies and mispaired species impurities but not the quality ranking of the clones.

Fungal siderophore biosynthesis catalysed by an iterative nonribosomal peptide synthetase.

Siderophores play a vital role in the viability of fungi and are essential for the virulence of many pathogenic fungal species. Despite their importance in fungal physiology and pathogenesis, the programming rule of siderophore assembly by fungal nonribosomal peptide synthetases (NRPSs) remains unresolved. Here, we report the characterization of the bimodular fungal NRPS, SidD, responsible for construction of the extracellular siderophore fusarinine C. The use of intact protein mass spectrometry, together with in vitro biochemical assays of native and dissected enzymes, provided snapshots of individual biosynthetic steps during NPRS catalysis. The adenylation and condensation domain of SidD can iteratively load and condense the amino acid building block cis-AMHO, respectively, to synthesize fusarinine C. Our study showcases the iterative programming features of fungal siderophore-producing NRPSs.

Complexity, cost, and content - three important factors for translation of clinical protein mass spectrometry tests, and the case for apolipoprotein C-III proteoform testing.

Complexity, cost, and content are three important factors that can impede translation of clinical protein mass spectrometry (MS) tests at a larger scale. Complexity stems from the many components/steps involved in bottom-up protein MS workflows, making them significantly more complicated than enzymatic immunoassays (EIA) that currently dominate clinical testing. This complexity inevitably leads to increased costs, which is detrimental in the price-competitive clinical marketplace. To successfully compete, new clinical protein MS tests need to offer something new and unique that EIAs cannot - a new content of proteoform detection. The preferred method for proteoform profiling is intact protein MS analysis, in which all proteins are measured as intact species thus allowing discovery of new proteoforms. To illustrate the importance of intact proteoform testing with MS and its potential clinical implications, we discuss here recent findings from multiple studies on the distribution of apolipoprotein C-III proteoforms and their correlations with key clinical measures of dyslipidemia. Such studies are only made possible with assays that are low in cost, avoid unnecessary complexity, and are unique in providing the content of proteoforms.

Liquid Extraction Surface Analysis (LESA) High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Mass Spectrometry for In Situ Analysis of Intact Proteins.

Liquid extraction surface analysis (LESA) is an ambient mass spectrometry technique which enables direct analysis of analytes from solid substrates. LESA is particularly suitable for the analysis of intact proteins from biological substrates such as thin tissue sections and bacterial colonies growing on agar. Nevertheless, these substrates present a challenge for LESA protein analysis owing to their inherent complexity. It is therefore beneficial to hyphenate LESA with a gas phase separation technique. One such technique is high-field asymmetric waveform ion mobility (FAIMS), in which ions are separated according to their different mobilities in high and low electric fields. Here, we describe the protocols for LESA FAIMS mass spectrometry of intact proteins in thin tissue sections and bacterial colonies.

An Improved Program for the Estimation of Protein Theoretical Relative Molecular Mass and Uncertainty.

Presented here is the National Institute of Standards and Technology (NIST) Mass Calculator, a software tool that provides both a relative molecular mass (Mr) value and an associated uncertainty value for a given protein sequence. The calculation of a protein Mr based on its amino acid residue sequence is performed by many software applications. Unfortunately, many of the results do not agree with each other. Reasons for this lack of consensus may include the use of rounded values, calculation errors, or alternative calculation methods, and it is often not possible to distinguish the source of the differences. Additionally, the result is provided without an estimate of precision. This would imply that the Mr is perfectly known when in fact it is not because the basis for the calculation, either the relative atomic masses or the standard atomic weights, are themselves provided as estimates. Until the release of the NIST Mass Calculator, there were no applications available to provide both an Mr value and an associated uncertainty value. Furthermore, the full disclosure of the computation methods and elemental values allows for independent verification of the results. The uncertainty is obtained via Monte Carlo simulation and is now a useful feature in light of the high resolution capability now available in the mass spectrometry of intact proteins.