Strategies to verify equimolar peptide release in mass spectrometry-based protein quantification exemplified for apolipoprotein(a).

Objectives: Quantitative protein mass spectrometry (MS) is ideally suited for precision diagnostics and for reference standardization of protein analytes. At the Leiden Apolipoprotein Reference Laboratory we apply MS strategies to obtain detailed insight into the protein-to-peptide conversion in order to verify that quantifier peptides are not partly concealed in miscleaved protein backbone.

Methods: Apolipoprotein(a) (apo(a)) was digested in a non-optimal manner to enhance the number of miscleaved peptides that were identified by high resolution liquid chromatography tandem-MS measurements.

Serum -Glycosylation RPLC-FD-MS Assay to Assess Colorectal Cancer Surgical Interventions.

A newly developed analytical strategy was applied to profile the total serum -glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was previously found that serum -glycome alterations in CRC were associated with patient survival. Here, fluorescent labeling of serum -glycans was applied using procainamide and followed by sialic acid derivatization specific for α2,6- and α2,3-linkage types via ethyl esterification and amidation, respectively.

Developing an SI-traceable Lp(a) reference measurement system: a pilgrimage to selective and accurate apo(a) quantification.

In the past decade a remarkable rebirth of serum/plasma lipoprotein(a) (Lp(a)) as an independent risk factor of cardiovascular disease (CVD) occurred. Updated evidence for a causal continuous association in different ethnic groups between Lp(a) concentrations and cardiovascular outcomes has been published in the latest European Atherosclerosis Society (EAS) Lp(a) consensus statement. Interest in measuring Lp(a) at least once in a person's lifetime moreover originates from the development of promising new Lp(a) lowering drugs.

Development of Tier 2 LC-MRM-MS protein quantification methods for liquid biopsies.

In the pursuit of personalized diagnostics and tailored treatments, quantitative protein tests contribute to a more precise definition of health and disease. The development of new quantitative protein tests should be driven by an unmet clinical need and performed in a collaborative effort that involves all stakeholders. With regard to the analytical part, mass spectrometry (MS)-based platforms are an excellent tool for quantification of specific proteins in body fluids, for example focused on cancer.

Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics.

Longitudinal Serum Protein Analysis of Women with a High Risk of Developing Breast Cancer Reveals Large Interpatient Versus Small Intrapatient Variations: First Results from the TESTBREAST Study.

The prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high-risk women. Serum samples were collected every (half) year up until diagnosis. Protein levels were longitudinally measured to determine intrapatient and interpatient variabilities.

Serum N-glycan profiles differ for various breast cancer subtypes.

Breast cancer is the most prevalent cancer in women. Early detection of this disease improves survival and therefore population screenings, based on mammography, are performed. However, the sensitivity of this screening modality is not optimal and new screening methods, such as blood tests, are being explored.

Clinical Perspective on Proteomic and Glycomic Biomarkers for Diagnosis, Prognosis, and Prediction of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is known as a highly aggressive malignant disease. Prognosis for patients is notoriously poor, despite improvements in surgical techniques and new (neo)adjuvant chemotherapy regimens. Early detection of PDAC may increase the overall survival.

The Time Has Come for Quantitative Protein Mass Spectrometry Tests That Target Unmet Clinical Needs.

Protein mass spectrometry (MS) is an enabling technology that is ideally suited for precision diagnostics. In contrast to immunoassays with indirect readouts, MS quantifications are multiplexed and include identification of proteoforms in a direct manner. Although widely used for routine measurements of drugs and metabolites, the number of clinical MS-based protein applications is limited.

Serum N-Glycome analysis reveals pancreatic cancer disease signatures.

Background &aims: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with loco-regional spread that makes the tumor surgically unresectable. Novel diagnostic tools are needed to improve detection of PDAC and increase patient survival. In this study we explore serum protein N-glycan profiles from PDAC patients with regard to their applicability to serve as a disease biomarker panel.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications.

Improved N- and C-Terminal Sequencing of Proteins by Combining Positive and Negative Ion MALDI In-Source Decay Mass Spectrometry.

The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above / 1000, while MALDI matrix ions generally hamper the detection of smaller singly charged fragments.

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution.

HILIC-MRM-MS for Linkage-Specific Separation of Sialylated Glycopeptides to Quantify Prostate-Specific Antigen Proteoforms.

Elevated serum prostate-specific antigen (PSA) levels in body fluids may indicate prostate cancer (PCa), but it is noted that the clinical performance is rather poor. Specificity and sensitivity values of 20 and 94% at a cutoff value of 4.1 ng/mL, respectively, result in overdiagnosis and unnecessary interventions.

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry.

Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization.

Dried blood spot N-glycome analysis by MALDI mass spectrometry.

Body fluid N-glycome analysis as well as glyco-proteoform profiling of existing protein biomarkers potentially provides a stratification layer additional to quantitative, diagnostic protein levels. For clinical omics applications, the collection of a dried blood spot (DBS) is increasingly pursued as an alternative to sampling milliliters of peripheral blood. Here we evaluate DBS cards as a blood collection strategy for protein N-glycosylation analysis aiming for high-throughput clinical applications.

Structural Analysis of Monoclonal Antibodies by Ultrahigh Resolution MALDI In-Source Decay FT-ICR Mass Spectrometry.

The emergence of complex protein therapeutics in general and monoclonal antibodies (mAbs) in particular have stimulated analytical chemists to develop new methods and strategies for their structural characterization. Mass spectrometry plays a key role in providing information on the primary amino acid sequence, post-translational modifications, and other structure characteristics that must be monitored during the manufacturing process and subsequent quality control assessment. In this study, we present a novel method that allows structural characterization of mAbs based on MALDI in-source decay (ISD) fragmentation, coupled with Fourier transform ion cyclotron resonance (FT-ICR) MS.

Automated Plasma Glycomics with Linkage-Specific Sialic Acid Esterification and Ultrahigh Resolution MS.

High-throughput mass spectrometry (MS) glycomics is an emerging field driven by technological advancements including sample preparation and data processing. Previously, we reported an automated protocol for the analysis of N-glycans released from plasma proteins that included sialic acid derivatization with linkage-specificity, namely, ethylation of α2,6-linked sialic acid residues and lactone formation of α2,3-linked sialic acids. In the current study, each step in this protocol was further optimized.

Proteoform Analysis to Fulfill Unmet Clinical Needs and Reach Global Standardization of Protein Measurands in Clinical Chemistry Proteomics.

In clinical testing of protein markers, structure variants of the measurand are often not taken into account. This heterogeneous character of protein measurands in immunoassays often renders test standardization impossible. Consequently, test results from different methods can lead to underdiagnosis or overdiagnosis and, thus, undertreatment or overtreatment of patients.

Serum -glycome alterations in colorectal cancer associate with survival.

Proteins are routinely measured in clinical laboratories for diagnosis, prognosis and therapy monitoring. Nevertheless, both test improvements (performance) and innovations (biomarkers) are needed, and protein -glycosylation offers a rich source of potential markers. Here, we have analyzed the total serum -glycome in a matched case-control study (124 cases versus 124 controls) of colorectal cancer patients.

Serum Protein -Glycosylation Changes with Rheumatoid Arthritis Disease Activity during and after Pregnancy.

Symptoms of rheumatoid arthritis (RA) improve during pregnancy, a phenomenon that was found to be associated with -glycosylation changes of immunoglobulin G. Recent advances in high-throughput glycosylation analysis allow the assessment of the -glycome of human sera as well. The aim of this study was to identify new protein -glycosylation properties that associate with changes in RA disease activity during and after pregnancy.

Structural Characterization of Biofunctionalized Gold Nanoparticles by Ultrahigh-Resolution Mass Spectrometry.

Biofunctionalized gold nanoparticles (AuNPs) enable innovative translational research and development in biomedicine. Biomolecules such as peptides, proteins, lipids, and carbohydrates can be assembled onto AuNPs to yield nanomaterials with unique properties for applications in imaging, photothermal therapy, vaccination strategies, and drug delivery. The characterization of functionalized AuNPs still remains an analytical challenge that normally requires the combination of multiple techniques.