Strategies to detect and quantify proteins in the clinical setting often employ enzyme-linked immunosorbent assays, or ELISAs. Although ELISAs are successfully used in many different settings, some clinically important proteins remain undetectable, especially in complex samples. ELISA testing is unable to distinguish between sequence variants or between target proteins with different post-translational modifications.1 Heterogeneous samples containing truncated isoforms of prostate specific antigen, for example, are difficult to accurately detect and can result in high false-positive rates.2
An alternative to ELISA is mass spectrometry (MS)-based targeted protein assays. Previous studies have demonstrated the reproducibility of MS in clinical settings3; however, low-abundance proteins — such as those found in plasma and serum — remain a challenge to detect using MS. In their 2013 publication, Krastins et al. reported their success in using an MS protocol specifically designed to identify 16 proteins that are clinically important and difficult to detect using traditional methods.4 Identification of proteins representing members of the apolipoprotein family, along with a wide range of abundance levels, was demonstrated. Medium- to high-abundance proteins identified in this study include ceruloplasmin, vitamin D binding protein, beta-2 microglobulin and C-reactive protein. Proteins with low-abundance levels identified in this study include procalcitonin, parathyroid hormone, insulin-like growth factor 1, prostate-specific antigen, erythropoietin, proprotein convertase subtilisin/kexin type 9 and amyloid beta. Collectively, the proteins were chosen to represent seven areas of interest, including neurology, Alzheimer’s disease, cardiovascular function, endocrine function, cancer and disease research.
For their experiments, mass spectrometric immunoassays were combined with selection reaction monitoring. A monolithic microcolumn activated with anti-protein antibodies fixed in a custom pipette tip (Thermo Scientific) was used with a Versette or Platemate Robotic workstation (Thermo Scientific). The high-throughput design allowed multiple protein samples to be quickly alkylated, then digested with trypsin in preparation for high-resolution liquid chromatography and tandem MS; however, smaller proteins could be analyzed without digestion with trypsin. Samples were then analyzed using an LTQ-Orbitrap XL (Thermo Scientific) mass spectrometer. Following the identification of variants, selection reaction monitoring was employed on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific).
This strategy involving mass spectrometric immunoassays and selection reaction monitoring, as described by Krastins et al., is highly sensitive and able to detect and quantify all proteins studied — including each specific protein isoform — in a matter of hours. Another advantage of this assay was its ability to detect several analytes simultaneously without hurting the sensitivity, unlike in clinical multiplexed ELISA. Because of these advantages, there is a very real possibility that this method could one day replace ELISA testing as the gold standard for protein detection in the clinical setting.
1. Nedelkov, D., et al. (2005) “Population proteomics: Addressing protein diversity in humans,” Expert Review of Proteomics, 2(3) (pp. 315–24).
2. Carter, H.B. (2012, May) “Differentiation of lethal and non lethal prostate cancer: PSA and PSA isoforms and kinetics,” Asian Journal of Andrology, 14(3) (355–60).
3. Prakash, A., et al. (2010) “Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays,” Journal of Proteome Research, 9(12) (pp. 6678–88).
4. Krastins, B., et al. (2013) “Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum,” Clinical Biochemistry, 6(6) (pp. 399–410).