Laboratory Testing Blog

Quantitative Analysis with Mass Spectrometric Detection No Longer Limited to Small Molecules

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Protein and peptide drugs cover a broad range of clinical application, such as bacterial infections, inflammatory and rheumatic diseases, pain, hypertension, stroke, AIDS and cancer.

Protein and peptide quantitation is most often performed using immunoassays.  Immunological methods can be highly sensitive and provide rapid analysis with high sample throughput, but a major disadvantage is the risk of cross-reactivity, as the used antibodies cannot discriminate between structurally related peptides.  In the cases of peptides and proteins, immunoassays would then quantify the total amount of related analytes.  Accurate quantification is therefore limited and data should be interpreted cautiously.  However, it is becoming more evident that quantitative analysis is no longer limited to small molecules.  Quantitative detection of large molecules can be readily performed using mass spectrometric techniques.

Liquid chromatography mass spectrometry (LC-MS) has become an indispensable tool in the analysis of complex protein mixtures. MS, with either ESI or MALDI, has great potential because it can differentiate between the various peptide analogs based on their collision-induced dissociation (CID) spectra or the protein isoforms based on their molecular weight or product-ion spectra.  The recombinant therapeutic proteins, protein vaccines and monoclonal antibodies can also be quantified by LC MS/MS analysis of specific peptides.

There are few examples of resent achievements accomplished in the field of absolute quantification of protein/peptide in biological fluids.  Some of these can be found in the following publications:

Enfuvirtide (T-20) and Tifuvirtide (T-1249) both belong to a novel class of antiretroviral agents for the treatment of Human Immunodeficiency Virus type-1 (HIV-1): the HIV-1 fusion inhibitors.  Enfuvirtide has successfully completed phase III clinical trials and is commercially available worldwide.  There are two articles that discuss this and can be found here and here.

An LC-MS/MS assay for the simultaneous quantification of two peptide HIV-1 fusion inhibitors, enfuvirtide and tifuvirtide and a metabolite of enfuvirtide (M-20) in human plasma has been successfully developed and validated using LC-MS/MS techniques.  The analytes were extracted from plasma by solid-phase extraction (SPE) on vinyl-copolymer cartridges.  The assay provided accurate and precise results and has demonstrated it usefulness in the analysis of plasma samples of HIV-1 infected patients.

An LC-MS/MS method has been developed and validated to allow separation and simultaneous quantification of the three human neutrophil HNP-1, -2, and -3 peptides in human plasma and serum.  You can find the reference article here.  The developed method also demonstrated applicability for quantitative measurement in other biological fluids, such as urine or saliva.  Therefore, the assay is likely to aid in the further exploration of the diagnostic value of these antimicrobial peptides and will firstly be applied to explore whether HNP-1, -2 and -3 play a role as serum markers of colon cancer.

Protein and peptide profiling in biological fluids of cancer patients has become an attractive approach in the search for novel cancer biomarkers.  However, little is known about the absolute concentration levels of these potential breast cancer biomarker peptides.  An LC–MS/MS method has been developed and validated for the quantification of multiple proteolytically derived peptide fragments with the potential to serve as biomarkers for breast cancer.  The assay allows simultaneous quantification of six potential breast cancer biomarker peptides at low level (0.4-10 ng/mL) detection.  You can read more about this by following this link.

Recently, as was discussed in are recearch article found that can be found here, Germany informed the European Commission's Early Warning and Response System (EWRS) of a significant increase in the number of patients with hemolytic uremic sysndrome (HUS) and bloody diarrhea caused by Escherichia coli (E. coli).  Investigations have now concluded that a Shiga toxin-producing Escherichia coli bacteria is responsible for this outbreak.  An announcement made on the afternoon of 11 June 2011 by the German authorities confirms that various types of sprouts produced by one farm south of the city of Hamburg are responsible for the E. coli outbreak in Germany.  Beginning in late May 2011, an outbreak of food-borne infections with a rare strain of the E. coli bacteria in Germany claimed at least 39 lives, unsettled the nation and threw European agriculture into disarray.

Escherichia coli is a Gram-negative bacterium of the family Enterobacteriacae.  It is relatively easy to cultivate, fast growing, and allows for feasible genetic manipulation.

Identification and quantitation of specific proteins has been achieved by using unique an LC-MS/MS technique.  The basic idea of this strategy is to optimize the detection of proteolytic peptides and to develop a sensitive and specific mass spectrometric assay.  In a first step, these assays are developed based on specific precursor/fragment ion pairs called MRM transitions as well as LC retention time information by analyzing synthesized peptides corresponding to a proteolytic protein fragment.  In a second step, proteins from real samples are identified and quantified by analyzing the real proteolytic peptides using the optimized MRM transitions.