Mass Spectrometry-Based Protein Biomarker Discovery and Measurement: Sensitivity is the Greatest Hurdle

Abstract

One of the greatest clinical needs in cancer diagnostics is the accurate detection of early-stage tumors that have not yet metastasized. Treatment of such early lesions is expected to have a direct impact on long-term survival. In contrast, once dissemination and organ colonization has begun, the effectiveness of all conventional therapy is limited. By definition, such early-stage lesions are very small (less than 0.5 cm) and often exist below the threshold of detection by sophisticated imaging such as MRI. Brown and Palmer [1] have estimated that a test for ovarian cancer would have to detect tumors smaller than 0.5 cm to achieve a 50% reduction in cancer mortality with an annual screen. Putative biomarkers elaborated into the circulation from a small pre-metastatic lesion will be diluted in the entire blood volume and can be subject to rapid clearance by a variety of mechanisms [2]. Thus, the expected concentration of a blood-borne diagnostic analyte derived from these crucially important, but tiny, clinical lesions will be much less than 1 ng/ml and will likely fall in the picograms-permilliliter range [1,2].Despite the rapid advancements in the application of mass spectrometry (MS) to biomarker discovery and quantitative measurement [3], serious physiologic challenges still remain that limit the sensitivity of MS. Consequently, the analytical sensitivity of MS for biomarker discovery is actually very low, particularly when applied to complex protein mixtures such as blood. Cancer-associated blood biomarkers exist within a sea of high-abundance proteins such as albumin and immunoglobulins. Moreover, circulating biomarkers are subjected to degradation during transportation and storage, further reducing their concentration. In part because of these physiologic limitations, the vast majority of hundreds of clinical analytes measured routinely in the clinical chemistry laboratory today cannot be detected by current MS discovery approaches [4]. MS is simply not sensitive enough to detect the typical clinical analyte that falls in the range of 50 pg/ml to 5 ng/ml. Established clinical immunoassays stand as tough competition because they are highly precise, reliable, and low in cost. In its current form, the routine sensitivity threshold MRM technology [3] is tens of nanograms per milliliter. The sensitivity of MRM needs to be improved 100-fold in order to compete with clinical immunoassays. This is a sobering reality for proponents of MS who hope it will replace conventional immunoassays.The majority of proteins cataloged by MS under the HUPO human plasma proteome project falls in the highabundance range [4]. Thus, a biomarker considered by MS to be negative in a sample may actually be present in the sample at a concentration below the level of MS sensitivity. A more sensitive technology such as protein array or ELISA may therefore detect a candidate biomarker in a control sample considered to be negative by MS. This should not be considered a false positive.Fortunately, advances in nanotechnology, and affinity capt...

Advances in human proteomics at high scale with the SOMAscan proteomics platform

“…In general, there remains a trade-off in analyzing complex biological samples, such as serum or plasma, between the comprehensiveness of the survey, the dynamic range or limits of detection or quantification, and absolute quantification [26,27]. Other historical challenges for MS proteomics can remain, including issues of reproducibility, throughput, and cost [8][9][10][11][12][13][14][15].…”

“…Metabolomics is today driven by machines we have understood for years -NMR and mass spectrometry (MS). Proteomics has been dominated by two technologies that are, so far, inadequate for deep unbiased proteomic measurements -enzyme-linked immuno-sorbent assays (ELISAs), based on antibodies, and MS. Large unbiased arrays of ELISAs do not scale to large numbers of analytes [5,7] while MS is difficult to do quantitatively and is best used for the most abundant proteins in a sample [8][9][10][11][12][13][14][15]. Thus, the so-called omics revolution refers to insights mostly gained from large-scale studies of nucleic acids, not proteins.…”

Slow Off-Rate Modified Aptamer Arrays for Biomarker Discovery and Diagnostic Applications

“…6 [also see Brody et al (2010); Zichi et al (2008)]. Furthermore, despite great advances and promise for mass spectrometry in clinical proteomics, many challenges remain, including issues of sensitivity (typically just under nM in current approaches), specificity, reproducibility, throughput, and cost (Addona et al 2009;Aebersold 2009;Bell et al 2009;Liotta and Petricoin 2010;Mitchell 2010;Pan et al 2009;Service 2008;Silberring and Ciborowski 2010). Nevertheless, we acknowledge that every MS parameter will improve over time.…”

The application of Aptamer in biomarker discovery

“…Unmatched sensitivity levels also hold back the application of MS in biomarker discovery, especially from body fluids. Putative biomarkers are subject to dilution and clearance after entering the circulation system, resulting in low concentrations of analysts ranging from 50pg/ml to 5ng/ml [ 12 , 13 ]. In contrast, the antibody-based method excels in both sensitivity and specificity, hence is extensively employed for the detection of a specific protein.…”