Mass Spectrometry Immunoassay (MSIA) Facility

Key System Highlights:

• Automated (robotic) protein biomarker validation
• Characterization of biotherapeutic drugs in clinical trials
• Coupled antibody affinity capture to mass spectrometry for quantitative and qualitative multiplexed immunoassays
• Distinguishes changes in proteins arising from amino acid substitutions, RNA splice variants, posttranslational modifications, proteolytic cleavge and bound molecules
• 500 samples/day throughput
• Initial menu of over 75 assays currently available
• Customized assays for other proteins defined by client

Contact Information:
Deanna L Streck
973-972-3170
streckdl@umdnj.edu

Background:
One of the major limitations of protein studies is the scarcity of high throughput technologies to simultaneous measure and readily recognize and characterize microheterogeneity that is encountered in human populations that can lead to disease. Mass spectrometry and database search engines are commonly used to identify proteolytic fragments that are purposely created as surrogates of the proteins in a sample. But in doing so, full-length protein information is lost during protolytic digestion thus eliminating the detection of unique protein characteristics within an individual stemming from genetic, transcriptional and posttranslational differences. Likewise, most conventional immunoassay approaches (e.g., ELISA and RIA) cannot differentiate structural changes, and are thus unable to recognize diversity and modifications at the protein level. The problem is further exacerbated when considering the dynamics of disease-states, where specific proteins are modulated, both quantitatively and qualitatively, within a disease population.

In 1995, Intrinsic Bioprobes Inc. (IBI) introduced the Mass Spectrometric Immunoassay (MSIA) as a technique for detecting and identifying peptides and proteins present in biological fluids [1]. This proprietary approach [2] is based on the isolation of analytes from biological milieu using immobilized antibodies, followed by release of the analytes and analysis using MALDI-TOF-MS. The original papers and patents describe methods for qualitative determination and identification of analytes, quantitative methodologies and multiplexed MSIA where multiple antibodies are used to target multiple analytes simultaneously. To simplify the approach IBI introduced the use of solid supports built into pipette tips for protein isolation prior to MALDI-TOF-MS (termed MSIA-Tips) [3]. These devices can address large volumes of biofluid and are used with 96-well (parallel) robotic workstations that can reach throughputs of ~ 500 assays/day [4].

After over a decade of industrial applications, IBI has recently made this technology available to academia; the Institute of Genomic Medicine is the currently the only medical center lab that has it's own high-throughput IBI MSIA system (since Feb 08). This system couples automated antibody affinity capture to mass spectrometry, allowing determination in one platform of both sensitive quantification with fmol sensitivity as well as qualitative changes in protein structure arising from amino acid substitutions, RNA splicing, posttranslational modifications, proteolytic processing and binding partners.  This technology has been well established in industry for over 10 years, used for biomarker validation and characterization as well as characterization of biotherapeutic drugs after they are given to patients in clinical trials.

The MSIA platform is particularly well suited for screening plasma and urinary proteins for microheterogeneity, and as such is a technique that fills the aforementioned "analytical void" existing between global proteomics approaches and conventional immunoassays. IBI has applied this technology to develop over 75 proprietary assays, used to target specific proteins for rigorous structural characterization and quantification.

An example of the resolution of this technology is exemplified in the two spectra provided by IBI showing intensity versus mass/charge ratio of the cardiovascular marker, creatine kinase (CK) as shown: (A) MSIA spectrum of CK from a healthy individual, and (B) MSIA of CK obtained from an individual suffering a myocardial infarction (MI). Briefly, both spectra were produced by repeatedly flowing 500 µL of plasma (diluted 1:1 with buffer) through affinity pipettes derivatized with polyclonal rabbit anti-human CK. After extraction and rinsing, retained proteins were eluted using acidified sinapinic acid MALDI matrix solution and analyzed using linear-DE-MALDI-TOF-MS. The spectra reveal signals from both the brain (CK-B; MW = 42,510) and muscle (CK-M; MW = 42,970) isoforms of the protein, which is consistent with the polyclonal antibody retrieving both isoforms (either individually or as part of a dimeric complex). Observed in MI-spectrum, however, are unique MI-variants of both the CK isoforms - as recognized by shifted signals at ~ 130 Da greater mass than the wild-type proteins. These MI-variants have been observed in MI-individuals but not in healthy individuals.

1. Nelson RW, Krone JR, Bieber AL, Williams P. 1995. Anal. Chem. 67: 1153-8.

2. The Mass Spectrometric Immunoassay (MSIA) process is patented under U.S. Patent #6,974,704, which is assigned with exclusive rights to Intrinsic Bioprobes, Inc. Tempe, AZ.

3. These devices and their use in HT-MSIA are protected under U.S. Patent #'s, 6,004,770; 6,093,541; 6,316,266; 6,498,039; 6,783,672; 7,083,723; 7,083,724; 7,087,163; 7,087,164; 7,087,165, all of which are assigned with exclusive rights to Intrinsic Bioprobes, Inc. Tempe, AZ.

4. Kiernan, U.A., Tubbs, K.A., Gruber, K., Nedelkov, D., Niederkofler, E.E., Williams, P., Nelson, R.W., "High Throughput Protein Characterization using Mass Spectrometric Immunoassay", Anal. Biochem . 301, 49 (2002).