Traditional protein immunoassays for clinical monitoring and diagnosis rely on specific antibody recognition and efficient workflows in a variety of different clinical samples. Unfortunately, in addition to factors such as matrix effects and low protein abundance affecting assay performance, antibody specificity often cannot distinguish between disease-specific isoforms relevant today for biomarker discovery.
However, mass spectrometry (MS) can not only distinguish between protein isoforms but also quantify their abundance, even at low levels. It also shows good inter-laboratory consistency, a vital requirement for clinical assay development. With advances in proteomics techniques, mass spectrometric immunoassays (MSIAs) are becoming more appropriate for clinical diagnostic sample handling.
Scientists at Thermo Scientific’s Biomarkers Research Initiatives in Mass Spectrometry Center (BRIMS) have collaborated with researchers from other institutions to develop and validate MS-based assays for clinically relevant proteins. Their combination of microcolumn immunoenrichment and MSIA with selected reaction monitoring (SRM) development and automated sample handling shows great potential in terms of accuracy and faster workflows. The system can prepare 96 samples in approximately four hours.
Krastins et al. (2013) present a paper outlining the rapid development of MSIA-SRM assays for 16 clinically relevant proteins; a complete listing of these proteins may be seen in Table 1 below.1 Their report includes comparison of results from the new assay methodology with those obtained from traditional clinical analyses measuring two proteins, parathyroid hormone (PTH) and insulin-like growth factor 1 (IGF1), in patient samples.
Table 1: Proteins assayed during development of MSIA-SRM methodology
ApoE |
Vitamin D binding protein |
Prostate-specific antigen |
ApoA1 |
Beta-2 microglobulin |
Erythropoietin |
ApoCI and ApoCIII |
C-reactive protein |
Proprotein convertase subtilisin/kexin type 9 |
ApoJ (clusterin) |
Procalcitonin |
Amyloid beta |
Ceruloplasmin |
Parathyroid hormone* |
Insulin-like growth factor 1* |
* Results compared with commercial immunoassay.
The teams used serum and plasma samples taken from patients with a number of clinical diagnoses: Alzheimer’s/neurology (n=40 individuals), cardiovascular/cerebrovascular (n=32), renal failure/endocrine abnormality/bone metabolism/low vitamin D (n=357), and growth disorders (n=289), along with serum pooled from a cohort of cancer patients.
Using 96 well microtiter plates and an automated pipetting workstation, samples were immunoenriched by elution through individual custom MSIA microcolumns (Thermo Scientific) each containing an antibody-activated monolithic matrix. Previous experiments determined antibody selection and optimal binding cycles before eluting the immunoenriched sample.
The authors comment that the repeated exposure to the antibody through multiple binding cycles reduced non-specific binding and increased the range of usable sample volumes. They also note that the antibody-based immunoenrichment step avoids depletion of albumin-bound biomarkers, as seen with other abundancy depletion techniques.
All samples, including quality controls and calibrating standards, underwent sample preparation as above and proteolytic digestion with either trypsin or another enzyme such as SV8 before liquid chromatography–tandem mass spectrometric (LC-MS/MS) analysis. Krastins et al. analyzed the samples with a high-resolution LTQ Orbitrap XL MS, identifying protein and peptide candidates suitable for SRM assay development with Proteome Discover software v1.3 (both Thermo Scientific). The researchers further optimized the individual SRM assays using Pinpoint software, v1.2 or 1.3, to select target peptides and proteolytic fragments from LC-MS/MS analysis of recombinant protein standards.
The scientists found that results obtained by SRM showed good symmetrical peaks, free from signal interference for all analytes. The limits of quanitification and detection closely matched the ranges expected in a clinical setting. The data showed good assay precision (%CV of triplicates ≤20%) and linearity with calibration curves (r2 ranged from 0.89 to 0.99).
Krastins and co-authors compared PTH (n=357 samples) and IGF1 (n=289) MSIA-SRM assay results with those obtained by commercial assay. Data correlated well: IGF1 r2=0.71; PTH r2 (all tryptic peptides)=0.87 and PTH r2 (N-terminal peptide)=0.67. Commenting on results for PTH, the researchers suggest that lack of antibody specificity in commercial assay might mean that SRM may give a more accurate measure of the active N-terminal peptide.
In conclusion, Krastins et al. consider their method to be a rapid, semi-automated alternative to traditional assays, with future potential for multiple analyte detection and quantification of disease-relevant biomarker isoforms.
For further information, see:
On-demand webinar (Bringing Mass Spectrometry into Your Laboratory) from Thermo Scientific, available at Improve Translational Proteomics Workflows Using MSIA-SRM Technology.
Reference
1. Krastins, B., et al. (2013) “Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins,” Clinical Biochemistry, 46(6) (pp. 399–410).
Leave a Reply