Mass spectrometry-based methods for protein biomarker quantification:On the road to clinical implementation

Proteins are compounds of which more than ten thousand different types can be found in our bodies, and measuring some of these proteins may, for example, be useful for detecting diseases. Numerous protein measurements are actually performed in hospitals and medical laboratories, although many diseases still lack appropriate tests to assist doctors in providing optimal patient care. Frank Klont conducted research that was focused on the development and simplification of mass spectrometry (MS-)based methods for measuring proteins in blood. MS is an analytical technique which allows for the separation of compounds based on their mass (weight) after which these compounds can be detected separately. Thereby, protein measurements by MS are considerably more specific as compared to those carried out with the analytical techniques that are mostly used in medical laboratories nowadays. MS, however, is not yet applied for patient care on a large scale, as MS instrumentation is rather difficult to operate and since procedures to prepare samples prior to MS measurements are fairly complex. In his thesis, Frank describes the development of an MS-based analytical platform for measuring a class of smaller proteins in an easy, reliable, and automated fashion. He furthermore presents a simple sample preparation methodology by which a class of challenging, low abundant proteins in blood can now be measured more easily by MS. Altogether, Frank attempted to simplify the process of developing novel MS-based blood tests for clinically-relevant proteins and thereby he aimed to lower threshold for implementing MS-based protein tests in medical laboratories

Mass spectrometry-based methods for protein biomarker quantification:On the road to clinical implementation

Proteins are compounds of which more than ten thousand different types can be found in our bodies, and measuring some of these proteins may, for example, be useful for detecting diseases. Numerous protein measurements are actually performed in hospitals and medical laboratories, although many diseases still lack appropriate tests to assist doctors in providing optimal patient care. Frank Klont conducted research that was focused on the development and simplification of mass spectrometry (MS-)based methods for measuring proteins in blood. MS is an analytical technique which allows for the separation of compounds based on their mass (weight) after which these compounds can be detected separately. Thereby, protein measurements by MS are considerably more specific as compared to those carried out with the analytical techniques that are mostly used in medical laboratories nowadays. MS, however, is not yet applied for patient care on a large scale, as MS instrumentation is rather difficult to operate and since procedures to prepare samples prior to MS measurements are fairly complex. In his thesis, Frank describes the development of an MS-based analytical platform for measuring a class of smaller proteins in an easy, reliable, and automated fashion. He furthermore presents a simple sample preparation methodology by which a class of challenging, low abundant proteins in blood can now be measured more easily by MS. Altogether, Frank attempted to simplify the process of developing novel MS-based blood tests for clinically-relevant proteins and thereby he aimed to lower threshold for implementing MS-based protein tests in medical laboratories

Assessment of sample preparation bias in mass spectrometry-based proteomics

For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data dependent acquisition, DDA) analyses using three different human head and neck tissues (i.e. nasal polyps, parotid gland, and palatine tonsils). Our study reveals differences between the sample preparation methods, for example with respect to protein and peptide losses, quantification variability, protocol-induced methionine oxidation and asparagine/glutamine deamidation as well as identification of cysteine containing peptides. However, none of the methods performed best for all types of tissues, which argues against the existence of a universal sample preparation method for proteome analysis

Quantification of surfactant protein D (SPD) in human serum by liquid chromatography-mass spectrometry (LC-MS)

Quantification of intact proteins in complex biological matrices by liquid chromatography-mass spectrometry (LC-MS) is a promising analytical strategy but is technically challenging, notably for concentrations at or below the ng/mL level. Therefore, MS-based protein quantification is mostly based on measuring protein-specific peptides, so-called 'surrogate peptides', that are released through proteolysis. While quantitative protein bioanalysis based on peptide LC-MS is much more sensitive, not every peptide is suitable in this respect. For example, some peptides are too small to be unique for a protein while others are too large to be measured with sufficient sensitivity, so careful selection of appropriate peptides is essential. Here we present a validated LC-MS method for quantification of surfactant protein D (SPD) at clinically relevant levels between 5 and 500 ng/mL using 50 mu L. of serum. This method targets two SPD-specific peptides in the C-type lectin, ligand binding domain of the SPD protein. One of these peptides contains a methionine residue which would typically be avoided because of its unstable nature. Some quantitative methods do target methionine-containing peptides, and corresponding workflows feature an oxidation step at the peptide level using hydrogen peroxide (H2O2) to convert all methionine residues to more stable methionine sulfoxides. For our method, such a procedure was associated with peptide loss, hence we developed an oxidation procedure at the protein level using H2O2 to oxidize methionine residues and the enzyme catalase to quench excess H2O2. This procedure may be applicable to other quantitative methods based on a surrogate peptide-based approach and may potentially also be useful for MS-based workflows targeting intact proteins

Adsorptive Microtiter Plates As Solid Supports in Affinity Purification Workflows

Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research