P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC–MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.     

Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential crosslinking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture crosslinking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine–lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein–protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how crosslinking MS will be an essential tool for understanding complex phenotypes.     

Quantification and Proteomic Characterization of β-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid β-oxidation, especially β-hydroxybutyrate, are fatty energy–supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine β-hydroxybutyrylation (Kbhb) is a β-hydroxybutyrate–mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.     

Uncovering post-translational modification-associated protein–protein interactions

In living systems, the chemical space and functional repertoire of proteins are dramatically expanded through the post-translational modification (PTM) of various amino acid residues. These modifications frequently trigger unique protein–protein interactions (PPIs) – for example with reader proteins that directly bind the modified amino acid residue – which leads to downstream functional outcomes. The modification of a protein can also perturb its PPI network indirectly, for example, through altering its conformation or subcellular localization. Uncovering the network of unique PTM-triggered PPIs is essential to fully understand the roles of an ever-expanding list of PTMs in our biology. In this review, we discuss established strategies and current challenges associated with this endeavor.     

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXY^R) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXY^R strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXY^R as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Pharmacokinetics of Sublingual Versus Oral Estradiol in Transgender Women

ObjectiveTo investigate the pharmacokinetics of 17β-estradiol (E2) administered orally versus those of 17β-E2 administered sublingually in transgender women.MethodsSingle doses of 17β-E2 were administered orally (1 mg) to 10 transgender women and then sublingually (1 mg) after a 1-week washout period. Blood samples were collected at baseline (0 hour) and at 1, 2, 3, 4, 6, and 8 hours after dosing. The samples were frozen and analyzed using liquid chromatography mass spectrometry (LC-MS/MS) and immunoassay.ResultsThe results demonstrated that sublingual E2 had a significantly higher peak serum E2 concentration of 144 pg/mL, measured using LC-MS/MS, compared with an oral E2 concentration of 35 pg/mL, measured using LC-MS/MS (P = .003). Sublingual E2 peaked at 1 hour and oral E2 peaked at 8 hours, as measured using LC-MS/MS. The area under the curve (AUC) (0-8 hours) for sublingual E2, measured using LC-MS/MS, was 1.8-fold higher than the AUC (0-8 hours) for oral E2, measured using LC-MS/MS. Additionally, sublingual E2 was found to have an increased E2-to-estrone ratio at all time points (1.1 ± 1.0 vs 0.7 ± 0.4, P ≤ .0001), the clinical significance of which is unclear.ConclusionOral E2 administered sublingually has a different pharmacokinetic profile, with higher serum E2 levels and AUC (0-8 hours) than traditionally administered oral E2. Multidaily dosing may be necessary to suppress testosterone levels with sublingual E2. The appropriate dosing, efficacy, and safety of sublingual E2, compared with those of other E2 preparations, are unknown.     

Widespread Arginine Phosphorylation in Staphylococcus aureus

Arginine phosphorylation was only recently discovered to play a significant and relevant role in the Gram-positive bacterium Bacillus subtilis. In addition, arginine phosphorylation was also detected in Staphylococcus aureus, suggesting a widespread role in bacteria. However, the large-scale analysis of protein phosphorylation, and especially those that involve a phosphoramidate bond, comes along with several challenges. The substoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis, and the acid instability of phosphoramidates was long thought to impede those enrichments. Furthermore, good spectral quality is required, which can be impeded by the presence of neutral losses of phosphoric acid upon higher energy collision–induced dissociation. Here we show that pArg is stable enough for commonly used Fe³⁺-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for the analysis of phosphopeptides. By profiling a serine/threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic arginine phosphorylated peptides, we validated the presence and localization of arginine phosphorylation in S. aureus. Finally, we could show that the knockdown of Stp1 significantly increases the overall amount of arginine phosphorylation in S. aureus. However, our analysis also shows that Stp1 is not a direct protein-arginine phosphatase but only indirectly influences the arginine phosphoproteome.     

Artifactual FA dimers mimic FAHFA signals in untargeted metabolomics pipelines

FA esters of hydroxy FAs (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Because of their low molar abundance, FAHFAs are typically quantified using highly sensitive LC-MS/MS methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact FA dimers formed in the LC-MS pipeline. These isobaric FA dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at subphysiological concentrations showed concentration-dependent formation of a diverse repertoire of FA dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species.  However, nonbiological FA dimers forming in the setting of high concentrations of FFAs can be misidentified as FAHFAs. Based on these results, we assembled a FA dimer database to identify nonbiological FA dimers in untargeted metabolomics datasets.     

Recent advancements in mass spectrometry–based tools to investigate newly synthesized proteins

Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry–based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging–based, and stable isotope labeling by amino acids in cell culture–based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.     

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.     

Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2^V617F and WT mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin β1 and to all nine potential glycosylation sites of integrin α2 were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in β1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin β1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and β1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F-mutated cells. Data are available via ProteomeXchange with identifier PXD030550.     

Quantitative Acetylomics Reveals Dynamics of Protein Lysine Acetylation in Mouse Livers During Aging and Upon the Treatment of Nicotinamide Mononucleotide

Lysine acetylation is a reversible and dynamic post-translational modification that plays vital roles in regulating multiple cellular processes including aging. However, acetylome-wide analysis in the aging process remains poorly studied in mammalian tissues. Nicotinamide adenine dinucleotide (NAD⁺), a hub metabolite, benefits health span at least in part due to the activation of Sirtuins, a family of NAD⁺-consuming deacetylases, indicating changes in acetylome. Here, we combine two antibodies for the enrichment of acetylated peptides and perform label-free quantitative acetylomic analysis of mouse livers during natural aging and upon the treatment of beta-nicotinamide mononucleotide (NMN), a NAD⁺ booster. Our study describes previously unknown acetylation sites and reveals the acetylome-wide dynamics with age as well as upon the treatment of NMN. We discover protein acetylation events as potential aging biomarkers. We demonstrate that the life-beneficial effect of NMN could be partially reflected by the changes in age-related protein acetylation. Our quantitative assessment indicates that NMN has mild effects on acetylation sites previously reported as substrates of Sirtuins. Collectively, our data analyze protein acetylation with age, laying critical foundation for the functional study of protein post-translational modification essential for healthy aging and perhaps disease conditions.     

Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at ?30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.     

inSPIRE: An Open-Source Tool for Increased Mass Spectrometry Identification Rates Using Prosit Spectral Prediction

Rescoring of mass spectrometry (MS) search results using spectral predictors can strongly increase peptide spectrum match (PSM) identification rates. This approach is particularly effective when aiming to search MS data against large databases, for example, when dealing with nonspecific cleavage in immunopeptidomics or inflation of the reference database for noncanonical peptide identification. Here, we present inSPIRE (in silico Spectral Predictor Informed REscoring), a flexible and performant open-source rescoring pipeline built on Prosit MS spectral prediction, which is compatible with common database search engines. inSPIRE allows large-scale rescoring with data from multiple MS search files, increases sensitivity to minor differences in amino acid residue position, and can be applied to various MS sample types, including tryptic proteome digestions and immunopeptidomes. inSPIRE boosts PSM identification rates in immunopeptidomics, leading to better performance than the original Prosit rescoring pipeline, as confirmed by benchmarking of inSPIRE performance on ground truth datasets. The integration of various features in the inSPIRE backbone further boosts the PSM identification in immunopeptidomics, with a potential benefit for the identification of noncanonical peptides.     

Relative Quantification of Proteins in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue Using Multiplexed Mass Spectrometry Assays

The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery.     

Proteome Remodeling of the Eye Lens at 50 Years Identified With Data-Independent Acquisition

The eye lens is responsible for focusing and transmitting light to the retina. The lens does this in the absence of organelles, yet maintains transparency for at least 5 decades before onset of age-related nuclear cataract (ARNC). It is hypothesized that oxidative stress contributes significantly to ARNC formation. It is in addition hypothesized that transparency is maintained by a microcirculation system that delivers antioxidants to the lens nucleus and exports small molecule waste. Common data-dependent acquisition methods are hindered by dynamic range of lens protein expression and provide limited context to age-related changes in the lens. In this study, we utilized data-independent acquisition mass spectrometry to analyze the urea-insoluble membrane protein fractions of 16 human lenses subdivided into three spatially distinct lens regions to characterize age-related changes, particularly concerning the lens microcirculation system and oxidative stress response. In this pilot cohort, we measured 4788 distinct protein groups, 46,681 peptides, and 7592 deamidated sequences, more than in any previous human lens data-dependent acquisition approach. Principally, we demonstrate that a significant proteome remodeling event occurs at approximately 50 years of age, resulting in metabolic preference for anaerobic glycolysis established with organelle degradation, decreased abundance of protein networks involved in calcium-dependent cell–cell contacts while retaining networks related to oxidative stress response. Furthermore, we identified multiple antioxidant transporter proteins not previously detected in the human lens and describe their spatiotemporal and age-related abundance changes. Finally, we demonstrate that aquaporin-5, among other proteins, is modified with age by post-translational modifications including deamidation and truncation. We suggest that the continued accumulation of each of these age-related outcomes in proteome remodeling contribute to decreased fiber cell permeability and result in ARNC formation.     

Development and validation of an isoform-independent monoclonal antibody–based ELISA for measurement of lipoprotein(a)

The study aims were to develop a new isoform-independent enzyme-linked immunoassay (ELISA) for the measurement of lipoprotein(a) [Lp(a)], validate its performance characteristics, and demonstrate its accuracy by comparison with the gold-standard ELISA method and an LC-MS/MS candidate reference method, both developed at the University of Washington. The principle of the new assay is the capture of Lp(a) with monoclonal antibody LPA4 primarily directed to an epitope in apolipoprotein(a) KIV₂ and its detection with monoclonal antibody LPA-KIV9 directed to a single antigenic site present on KIV₉. Validation studies were performed following the guidelines of the Clinical Laboratory Improvement Amendments and the College of American Pathologists. The analytical measuring range of the LPA4/LPA-KIV9 ELISA is 0.27–1,402 nmol/L, and the method meets stringent criteria for precision, linearity, spike and recovery, dilutability, comparison of plasma versus serum, and accuracy. Method comparison with both the gold-standard ELISA and the LC-MS/MS method performed in 64 samples with known apolipoprotein(a) isoforms resulted in excellent correlation with both methods (r=0.987 and r=0.976, respectively). Additionally, the variation in apolipoprotein(a) size accounted for only 0.2% and 2.2% of the bias variation, respectively, indicating that the LPA4/LPA-KIV9 ELISA is not affected by apolipoprotein(a) size polymorphism. Peptide mapping and competition experiments demonstrated that the measuring monoclonal antibodies used in the gold-standard ELISA (a-40) and in the newly developed ELISA (LPA-KIV9) are directed to the same epitope, ⁴⁰⁷⁶LETPTVV⁴⁰⁸², on KIV₉. In conclusion, no statistically or clinically significant bias was observed between Lp(a) measurements obtained by the LPA4/LPA-KIV9 ELISA and those obtained by the gold-standard ELISA or LC-MS/MS, and therefore, the methods are considered equivalent.