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Jun 15, 2012 (Vol. 32, No. 12)

Mass Spec Gives Shot in Arm to Proteomic Analysis

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    Researchers at Erasmus Medical Center are using SRM, which exploits the unique capabilities of triple quadrupole mass spectrometers in their pre-eclampsia research. [Waters]

    Mass spectrometry (MS) is firmly established as a technique of choice for unraveling structural characteristics of chemical compounds. However, until recently, detection of low abundant peptide and protein analytes remained challenging because of poor efficiencies in ion formation and transmission.

    The recent “American Society For Mass Spectrometry” conference was largely dedicated to the changing role of MS in proteomics analysis. Recent developments in instrument design have led to lower limits of detection, and better understanding of gas-phase chemistry has opened possibilities for more sophisticated peptide and protein analysis.

    The technologies featured in this article are only a few of the conference highlights underscoring the recent and anticipated developments in MS and related techniques applied to the analysis of dynamic proteomes.

    Consistent identification and quantification of target peptides, especially those present in low abundance, is essential for diagnosis and prognosis of diseases. This level of precision is not quite achievable by current proteomic technologies. A new MS-based approach, termed selective reaction monitoring (SRM), is emerging as a technology that has a unique potential for reliable quantification of low abundance analytes.

    SRM exploits the unique capabilities of triple quadrupole mass spectrometers. A quadrupole is a component of the instrument responsible for filtering sample ions, based on their mass-to-charge ratio. For SRM, three quadrupoles are deployed, with the first and the third components acting as mass-charge filters, and the second acting as a collision cell.

    “For each target protein we select several peptides that uniquely define the target in the MS profile,” said Theo Luider, Ph.D., head of the laboratories of neuro-oncology, department of neurology, Erasmus Medical Center.

    “Next, these peptides are fragmented and fragment ions are separated by mass and charge again. This process creates a set of unique transitions from peptide to fragment for each peptide. Monitoring three to four transitions for each target peptide accurately quantifies any target peptide in the cellular proteome in a reproducible manner.”

    By using SRM, the team was able to quantify two selected fragments of calcyclin, a putative biomarker for pre-eclampsia. Although the case of pre-eclampsia, a life-threatening condition associated with 8% of pregnancies, is not yet understood, its onset was associated with increased transcription of certain placental proteins.

    Dr. Luider and his colleagues used laser microdissected materials from paraffin-embedded placental tissue.

    “We used stable-isotope labeled analogs of target peptides to spike the samples,” continued Dr. Luider. “These analogs are chemically identical to native peptides; they behave identically on the separation columns and produce identical transitions. Comparison with such internal reference standards confirms the recovery of calcyclin peptides and determines their absolute amounts in the sample.”

    The most recent study quantified calcyclin in serum samples. Endogeonus levels of calcyclin were detectable in crude serum, but fractionation on the cation-exchange column significantly improved signal detection. “Now that we have improved the method, we would like to validate the diagnostic potential of SRM technology using larger patient cohorts,” added Dr. Luider.

  • Enhancing Biomarker Discovery

    “SRM delivers a unique fragment ion that can be monitored and quantified in the midst of a very complicated matrix,” said Melissa Radabaugh, senior R&D scientist, Sigma-Aldrich. “However, to achieve the desired result, the column, ionization source, and the flow rate need to be optimized.”

    Previously, while at Pfizer, Radabaugh and her team greatly improved the sensitivity of detection of nitrotyrosine, a marker of many pathological conditions resulting from oxidative/nitrative cell damage, by developing an on-line immunoaffinity SRM LC-MS/MS method. Optimization of the process parameters enabled detection of nitrotyrosine in the low picomolar levels. Coupling with an antibody column further improved the specificity of detection.

    The assay was tested on a variety of biological fluids including urine, plasma, and cerebrospinal fluid, ensuring that the method is robust enough to support evaluation of nitric oxide-driven pathologies and their response to treatment.

    Radabaugh’s most recent study optimized a system for quantification of peptides. In order to translate these studies into clinic-based diagnostics, the serum samples were spiked with a nonhuman synthetic peptide. These defined standard mixtures were used for optimization of liquid chromatography (LC) and MS parameters.

    The team tested various separation parameters such as flow rates, peak shape, retention time, and delay-time volumes using several separation columns. The goal was to achieve the optimal separation using capillary flow rates as opposed to a more commonly used nanoflow. In the context of analyzing proteins by SRM, capillary flow has a number of advantages. This flow rate prevents issues of clogging when complex protein mixtures are used.

    The column with the best outcomes was coupled with different ionization sources. The ion source is the first part of the mass spectrometer and is used to ionize the sample. Under the testing conditions, the Michrom Advance source demonstrated the strongest signal. As a whole, the system was able to uptake a larger amount of sample, making this a useful system for biomarker discovery, quantitation, and validation.

    “The advantages of microflow includes the ability to load a larger sample and to achieve better separation,” said Tina Settineri, Ph.D., director, HPLC products, Eksigent division of AB Sciex.

    “However, nanoflow provides the absolute best sensitivity for mass spectrometry, because the sample becomes highly concentrated. For added flexibility between nanoflow and microflow LC, nanoLC customers can effortlessly convert between nano- and microflow by interchanging the flow channels in our 2D nanoLC systems, such as the NanoLC-Ultra® 2Dplus system. Furthermore, Eksigent’s ekspert™ microLC 200 system enables two different flow-rate ranges.”

    The most recent enhancement coupled the nanoflow liquid chromatography with high-performance mass spectrometry to achieve the best possible combination of high sensitivity and high acquisition speeds. This combination of hardware is essential to deal with samples of high complexity, such as serum or cellular digests.

    “The key to increasing protein separation on the nanoLC was to increase the column length,” continued Dr. Settineri. “This in turn increased the separation time, and hence the time available for MS/MS acquisition, resulting in the most proteins identified utilizing just under 50 MS/MS scans per second. By going from a 15 cm column to a 30 cm column, we were able to increase the number of identified proteins by 20%, while keeping the identification errors to less than 1%.”

    The company is exploring ways to further increase column length to 50 cm in order to extend coverage to a more complete proteome. The analysis was performed on the TripleTOF™5600 system, a top of the line hybrid quadrupole time of flight (TOF) mass spectrometer.

    This instrument is a central piece of the proteomic technique that quantifies nearly all proteins in a sample in a single analysis. SWATH™ Acquisition does not simply detect a single precursor ion. Instead, it uses a certain predefined mass range to perform MS/MS acquisition. SWATH technique generates highly specific fragment ion data for all peptides within this mass range.

    Resulting fragment ion chromatograms uniquely identify each peptide of interest, just like in SRM. The technique provides a complete quantitative profile of all proteins in the sample.

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