Irene van den Broek

Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry

With the growing interest for peptides and proteins in different kinds of fields, e.g. pharmacy, clinical diagnostics or food industry, the quantification of these compounds is becoming more and more important. Quantitative analysis of these analytes in biological matrices, however, remains a challenging task, due to the complexity of both the matrix and the analytical characteristics of these large bio-molecules. Liquid chromatography coupled to (tandem) mass spectrometry (LC-MS or LC-MS/MS) is the preferred analytical technique for peptide analysis as it allows very selective and sensitive measurements. This article summarizes the numerous published LC-MS applications for the quantification of peptides in biological matrices and discusses all different issues herewith concerned. This includes chromatographic aspects as the selection and effects of mobile and stationary phase, flow rate and temperature, as well as mass spectrometric characteristics such as ionization and detection modes, collision-induced dissociation of peptides and factors influencing the mass spectrometric response. For both techniques the main properties of all described methods have been listed, creating a comprehensive overview with the peptide analytes divided into different classes. Likewise, all other issues concerned with quantitative bioanalysis have been evaluated in detail, including extensive consideration of several different applied sample pre-treatment techniques and reflection of subjects as the choice for an internal standard and assay validation. Furthermore, several issues which are of particular interest for the quantitative bioanalysis of peptide compounds like peptide adsorption and degradation have been regarded.

Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry.

An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The peptides include bradykinin, Hyp(3)-bradykinin, des-Arg(9)-bradykinin and fragments of fibrinogen alpha-chain (Fib-alpha([605-629])), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH(4[666-687])) and complement component 4a (C4a([1337-1350])). Ile(13)-ITIH(4[666-687]), d20-C4a([1337-1350]) and Sar-D-Phe(8)-des-Arg(9)-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM D-L-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10-500 ng/ml), Hyp(3)-bradykinin (4-200 ng/ml), des-Arg(9)-bradykinin (2-100 ng/ml), Fib-alpha([605-629]) (120-3000 ng/ml), ITIH(4[666-687]) (0.4-10 ng/ml) and C4a([1337-1350]) (1-25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.

Serum Degradome Markers for the Detection of Breast Cancer

Many proteins have been proposed as potential biomarkers for breast cancer. Yet, validation of their discriminative value using quantitative methods has scarcely been performed. In this study, we investigated the discriminative value of six peptides that were previously proposed to be generated by breast cancer specific exoproteases: bradykinin, des-Arg(9)-bradykinin, Hyp(3)-bradykinin, and fragments of fibrinogen alpha-chain (Fib-alpha ([605-629])), complement component 4a (C4a ([1337-1350])), and interalpha trypsin inhibitor heavy chain 4 (ITIH4 ([666-687])). Their absolute serum concentrations were measured with a completely validated liquid chromatography-tandem mass spectrometric assay (LC-MS/MS) and compared between 62 newly diagnosed breast cancer patients and 62 controls matched for age and sample storage duration. Both ITIH4 ([666-687]) and des-Arg(9)-bradykinin showed statistically significantly higher median concentrations in breast cancer samples than in matched control samples. Additionally, we analyzed serum samples collected after surgical removal of the tumor, in which median ITIH4 ([666-687]) and des-Arg(9)-bradykinin concentrations were significantly decreased and not statistically significantly different from concentrations in the controls anymore. In a combined analysis, ITIH4 (666-687]) and des-Arg(9)-bradykinin independently contributed to the discrimination between cases and controls. In this study, we confirmed that the exoprotease breakdown peptides, ITIH4 (666-687]) and des-Arg(9)-bradykinin, differed between breast cancer cases and controls, supporting the potential of degradome markers for the diagnosis of breast cancer.

Automated Multiplex LC-MS/MS Assay for Quantifying Serum Apolipoproteins A-I, B, C-I, C-II, C-III, and E with Qualitative Apolipoprotein E Phenotyping

BACKGROUND Direct and calculated measures of lipoprotein fractions for cardiovascular risk assessment suffer from analytical inaccuracy in certain dyslipidemic and pathological states, most commonly hypertriglyceridemia. LC-MS/MS has proven suitable for multiplexed quantification and phenotyping of apolipoproteins. We developed and provisionally validated an automated assay for quantification of apolipoprotein (apo) A-I, B, C-I, C-II, C-III, and E and simultaneous qualitative assessment of apoE phenotypes. METHODS We used 5 value-assigned human serum pools for external calibration. Serum proteins were denatured, reduced, and alkylated according to standard mass spectrometry-based proteomics procedures. After trypsin digestion, peptides were analyzed by LC-MS/MS. For each peptide, we measured 2 transitions. We compared LC-MS/MS results to those obtained by an immunoturbidimetric assay or ELISA. RESULTS Intraassay CVs were 2.3%-5.5%, and total CVs were 2.5%-5.9%. The LC-MS/MS assay correlated (R = 0.975-0.995) with immunoturbidimetric assays with Conformité Européenne marking for apoA-I, apoB, apoC-II, apoC-III, and apoE in normotriglyceridemic (n = 54) and hypertriglyceridemic (n = 46) sera. Results were interchangeable for apoA-I ≤3.0 g/L (Deming slope 1.014) and for apoB-100 ≤1.8 g/L (Deming slope 1.016) and were traceable to higher-order standards. CONCLUSIONS The multiplex format provides an opportunity for new diagnostic and pathophysiologic insights into types of dyslipidemia and allows a more personalized approach for diagnosis and treatment of lipid abnormalities.

Quantifying protein measurands by peptide measurements: where do errors arise?

Clinically actionable quantification of protein biomarkers by mass spectrometry (MS) requires analytical performance in concordance with quality specifications for diagnostic tests. Laboratory-developed tests should, therefore, be validated in accordance with EN ISO 15189:2012 guidelines for medical laboratories to demonstrate competence and traceability along the entire workflow, including the selected standardization strategy and the phases before, during, and after proteolysis. In this study, bias and imprecision of a previously developed MS method for quantification of serum apolipoproteins A-I (Apo A-I) and B (Apo B) were thoroughly validated according to Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2 and EP09-A3, using 100 patient sera and either stable-isotope labeled (SIL) peptides or SIL-Apo A-I as internal standard. The systematic overview of error components assigned sample preparation before the first 4 h of proteolysis as major source (∼85%) of within-sample imprecision without external calibration. No improvement in imprecision was observed with the use of SIL-Apo A-I instead of SIL-peptides. On the contrary, when the use of SIL-Apo A-I was combined with external calibration, imprecision improved significantly (from ∼9% to ∼6%) as a result of the normalization for matrix effects on linearity. A between-sample validation of bias in 100 patient sera further supported the presence of matrix effects on digestion completeness and additionally demonstrated specimen-specific biases associated with modified peptide sequences or alterations in protease activity. In conclusion, the presented overview of bias and imprecision components contributes to a better understanding of the sources of errors in MS-based protein quantification and provides valuable recommendations to assess and control analytical quality in concordance with the requirements for clinical use.

Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA-MALDI-TOF-MS workflow.

A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.

Evaluation of interspecimen trypsin digestion efficiency prior to multiple reaction monitoring-based absolute protein quantification with native protein calibrators.

Implementation of quantitative clinical chemistry proteomics (qCCP) requires targeted proteomics approaches, usually involving bottom-up multiple reaction monitoring-mass spectrometry (MRM-MS) with stable-isotope labeled standard (SIS) peptides, to move toward more accurate measurements. Two aspects of qCCP that deserve special attention are (1) proper calibration and (2) the assurance of consistent digestion. Here, we describe the evaluation of tryptic digestion efficiency by monitoring various signature peptides, missed cleavages, and modifications during proteolysis of apolipoprotein A-I and B in normo- and hypertriglyceridemic specimens. Absolute quantification of apolipoprotein A-I and B was performed by LC-MRM-MS with SIS peptide internal standards at two time points (4 and 20 h), using three native protein calibrators. Comparison with an immunoturbidimetric assay revealed recoveries of 99.4 ± 6.5% for apolipoprotein A-I and 102.6 ± 7.2% for apolipoprotein B after 4 h of trypsin digestion. Protein recoveries after 20 h trypsin incubation equaled 95.9 ± 6.9% and 106.0 ± 10.0% for apolipoproteins A-I and B, respectively. In conclusion, the use of metrologically traceable, native protein calibrators looks promising for accurate quantification of apolipoprotein A-I and B. Selection of rapidly formed peptides, that is, with no or minor missed cleavages, and the use of short trypsin incubation times for these efficiently cleaved peptides are likely to further reduce the variability introduced by trypsin digestion and to improve the traceability of test results to reach the desirable analytical performance for clinical chemistry application.

The absolute quantification of eight inter‐α‐trypsin inhibitor heavy chain 4 (ITIH4)‐derived peptides in serum from breast cancer patients

Purpose: Various studies exploring the potential of the low‐molecular‐weight serum peptidome have identified proteolytic cleavage products of inter‐α‐trypsin inhibitor heavy chain‐4 (ITIH4) as potential markers for different types of cancer, presumably generated by tumor‐associated exoproteases. However, further elucidation of the discriminative properties of such peptides requires specific quantitative analytical methods.

Metrological traceability in mass spectrometry-based targeted protein quantitation: A proof-of-principle study for serum apolipoproteins A-I and B100

UNLABELLED In this study, we have followed up on previous liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometry (MS) approaches for measurement of apolipoprotein (apo) A-I and apo B100 in serum aiming for implementation of a multiplexed assay in a clinical chemistry laboratory with full metrological traceability. Signature peptides were selected and detected by dynamic MRM, and stable isotope labeled (SIL)-peptides were used as internal standards. Five apo A-I and four apo B100 peptides were measured in serum digests with linearity (R(2)>0.992) in the physiologically relevant concentration ranges. Linearity with regard to protein concentration was ascertained at five concentration levels (R(2)>0.926 and R(2)>0.965, for the apo A-I and apo B100 peptides, respectively). Three native value-assigned sera were used as external calibrators for further method verification. Imprecision values on sample preparation and LC-MS/MS acquisition were below the established minimal specifications for apo A-I and apo B100 (5.0% and 5.3%, respectively). Correlation of LC-MS/MS results with immunoturbidimetric assay results, for normo- and hypertriglyceridemic samples, showed R(2)>0.944 for apo A-I and R(2)>0.964 for apo B100. This LC-MS/MS method has potential for clinical application in normo- and dyslipidemic patients. BIOLOGICAL SIGNIFICANCE Measurement of apo A-I and apo B100 may offer an alternative to high and low density lipoprotein cholesterol (HDL-c and LDL-c) methods for cardiovascular disease risk assessment in dyslipidemic patients [1]. An LC-MS/MS method for apo A-I and apo B100 has the advantage of antibody independent and specific detection of protein signature peptides. The introduction of an LC-MS/MS method for apo A-I and apo B100 can serve as an example for many existing and newly developed (multiplex) biomarker methods in quantitative clinical chemistry proteomics (qCCP). Such LC-MS/MS methods should meet basic clinical chemistry principles with regard to test evaluation [2]. Criteria for imprecision should be pre-defined, e.g., based on biological variation. The use of commutable and traceable serum-based calibrators will improve inter-laboratory reproducibility of LC-MS/MS methods and may contribute to a more rapid transition of biomarker discovery to clinical utility with benefit for the patient treatment and improvement of general health care.

Sensitive liquid chromatography/tandem mass spectrometry assay for absolute quantification of ITIH4-derived putative biomarker peptides in clinical serum samples

To explore the potential of peptide fragments derived from inter-alpha-trypsin inhibitor heavy chain-4 (ITIH(4)) as serum markers for different cancer types, sensitive and specific analytical assays are required. Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) would be suitable; however, a previously developed method for quantification of eight ITIH(4) fragments (ITIH(4)-21, -22, -25, -26, -27, -28, -29 and -30) was found to be insensitive for clinical use. A more sensitive LC/MS/MS assay has now been developed and validated, which was further optimized to facilitate analyses of large sets of clinical serum samples. Benefits compared to the previous method include reduction of sample volume (100 microL), omission of protein precipitation and evaporation and transferring solid-phase extraction (SPE) to a 96-well format. Chromatographic separation on an XBridge BEH300 C(18) column, using a water/methanol gradient containing acetic acid, was coupled to triple quadrupole mass spectrometric detection, applying heated electrospray ionization. Method validation revealed deviations from nominal concentrations below 10.1% and intra- and inter-assay precisions below 17.4 and 20.0%, respectively, at the lower limit of quantification (LLOQ) for all peptides. The reported changes resulted in more rapid and efficient analyses and reduced LLOQs for the six less abundant peptides (1.2; 1.0; 1.2; 2.0; 2.0 and 2.0 ng/mL vs. 2.1; 2.0; 2.5; 2.6; 2.2 and 2.4 ng/mL for ITIH(4)-21, -22, -25, -27, -28 and -29, respectively). The method has shown its applicability by quantifying all peptides in appropriate concentration ranges in serum from healthy volunteers and application to clinical samples from breast cancer patients.