General Mass Spectrometry Assay Using Continuously Eluting Co-Fractionating Reporters of Mass Spectrometry Detection Efficiency

a mass spectrometry and reporter technology, applied in the field of mass spectrometry, can solve the problems of ion suppression or enhancement, adversely affecting the ability to assay or measure chemical and biological analytes, and affecting the amount of charged analytes that reach the detector of the mass spectrometer

Inactive Publication Date: 2014-09-11
UAB RES FOUND
View PDF0 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for measuring the amount of analytes in samples using reporters that co-fractionate with the analytes during gel electrophoresis and liquid chromatography. These reporters include individual reporters peptides or artificial proteins that generate distinct peaks during mass spectrometry. The methods involve combining the reporters with the sample containing the analyte, subjecting the mixture to fractionation by liquid chromatography, and detecting the analyte using a mass spectrometric technique. The invention also provides compositions and methods for designing chemical or biological reporters with peak-to-peak adjacent elution ranges for organic and biological compounds. The technical effects of the invention include improved accuracy in measuring analyte concentrations and the ability to normalize for sample recovery and ion suppression or enhancement.

Problems solved by technology

Although, MS does have issues that can adversely affect the ability to assay or measure chemical and biological analytes.
One problematic issue is ion suppression or enhancement in electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix assisted laser desorption ionization (MALDI) that are caused by nonvolatile compounds including salts, ion-pairing agents, endogenous compounds, and co-eluting compounds present in samples being evaluated by mass spectrometry.
These ion suppressing or enhancing factors, referred to as sample matrix, cause changes in the efficiency of droplet formation or evaporation during the ionization process and ultimately result in an altered amount of charged analytes that reach the detector of the mass spectrometer.
Despite SAM's widespread use for targeted measurements of specific compounds, it cannot be easily applied to proteomics or high-throughput experiments because investigators often lack a priori knowledge of what they wish to measure, purchasing standards for all the peptides of interest is prohibitively expensive, or the requirement of doing multiple measurements using the SAM is too costly.
The drawbacks to AQUA peptides include the necessity of having to purchase a separate AQUA peptide(s) for each protein to be measured from a biological sample, in some cases without experimental knowledge of the specific peptides mass spectrometry observability including limit of detection and limit of quantitation values.
These artificial proteins differ from the proposed technology because of their limited elution range, which results in these proteins not being generally applicable to correct for matrix effects.
The main drawback to using the QCAL1 peptides is that the amount of ion suppression or enhancement can vary dramatically throughout an LC / MS run (Stahnke, H, et al J Anal Chem, 2009; 81: 2185-2192).
Postcolumn infusion has the drawback of requiring additional pumps or gradient formers and reduced sensitivity caused by dilution of the sample with the postcolumn infusate.
The interpretation of data is difficult because each proteolytic peptide containing heavy or light amino acids generated from different samples are combined prior to mass spectrometric analysis resulting in different molecular ions and fragment ions depending upon the number of heavy or light amino acids in each peptide (McIlwain, S, et al Bioinformatics, 2008; 24: 339-347).
For instance, the metabolic conversion of arginine to proline in eukaryotes has been documented in SILAC experiments which causes incorrect quantitation by MS (Hwang, S I, et al Mol Cell Proteomics, 2006; 5: 1131-1145) and because more than 50% of tryptic peptides in large data sets contain proline (Van Hoof, D, et al Nat Methods, 2007; 4: 677-678) this is a significant problem for quantitative proteomic measurements.
The final drawback for SILAC is that the methodology cannot be used to study tissue samples from animal and human sources.
The drawbacks with iTRAQ include variability among samples caused by the extra steps involved in labeling with the iTRAQ reagent.
Also, the sensitivity and quantitative capability with iTRAQ is greatly reduced due to the combining of samples and corresponding increase in sample complexity.
This increase in sample complexity often necessitates analyzing samples multiple times by mass spectrometry and generating an exclusion list of the most abundant ions.
If the labeling reactions are incomplete, multiple products will be generated which complicate the quantitative analysis.
In addition, iTRAQ is limited in utility to mass spectrometric instruments which have the ability to measure a specific precursor ion followed by fragmentation of the precursor ion commonly referred to as MS / MS.
Samples to be compared are individually reduced, denatured, and labeled potentially providing a source of differential error prior to mixing the different samples.
In addition to the possible variability introduced by the steps involved in labeling each individual sample, ICAT has the drawback of labeling only cysteine containing peptides which limits analysis to only a small fraction of sample peptides.
A major drawback for the label free methods are that there is no compensation for differences in the suppression or enhancement of peptides caused by the variable makeup of different sample matrix components.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • General Mass Spectrometry Assay Using Continuously Eluting Co-Fractionating Reporters of Mass Spectrometry Detection Efficiency
  • General Mass Spectrometry Assay Using Continuously Eluting Co-Fractionating Reporters of Mass Spectrometry Detection Efficiency
  • General Mass Spectrometry Assay Using Continuously Eluting Co-Fractionating Reporters of Mass Spectrometry Detection Efficiency

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0149]An example of using a chemical reporter set to measure the absolute level of ranitidine in human plasma. An inventive set of chemical reporters of mass spectrometry detection efficiency were created based on their predicted ability to elute in a continuous peak to peak manner during a portion of an LC separation. The HPLC retention index (R.I.) of salbutamol (R.I. 225), 7-methylxanthine (R.I. 225), 4-aminoantipyrine (R.I. 226), famotidine (R.I. 228), DL-4-hydroxy-3-methoxymandelic acid (R.I. 230), and trans-2-phenylcyclopropylamine hydrochloride (R.I. 232) were previously described in Hill, D W, et al J Anal Tox, 1994; 18: 233-242. These chemical reporters were dissolved in 5% formic acid and mixed in said concentrations: salbutamol (2 nanograms / uL), 7-methylxanthine (12 nanograms / uL), 4-aminoantipyrine (10 nanograms / uL), famotidine (200 nanograms / uL), DL-4-hydroxy-3-methoxymandelic acid (18 nanograms / uL), trans-2-phenylcyclopropylamine hydrochloride (10 nanograms / uL). A 5 nan...

example 2

[0151]Design of artificial sequences: An in silico set of 20 artificial reporter peptides were created that were predicted by the Sequence Specific Retention Calculator Version 3.0 to elute in a peak to peak fashion during a fraction of the entire liquid chromatography separation with a 2% acetonitrile / minute linear gradient. These peptide sequences are shown in FIG. 7. and were checked to ensure that they do not exist in sequenced genomes by using the Prowl software program which searches sequenced genomes from the NCBI database (last updated on May 16, 2010) to ensure that the inventive peptides would not be generated by trypsin hydrolysis of proteins from the organisms of sequenced genomes. The peptides shown in FIG. 7. were assembled into an artificial protein and created by expression of the cDNA sequence shown in FIG. 8. This cDNA sequence was synthesized and subcloned into the NdeI and XhoI sites of the pET23b+ expression vector. A nucleotide sequence for protein expression o...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides general methods for quantifying any conceivable compound including small organic molecules and biological molecules in mass spectrometric measurements. The methods include the use of chemical or biological reporters such as artificial polypeptides containing proteolytic cleavage sites, which provide proteolytic reporter peptides for standardization of mass spectrometric detection efficiency. In addition to mass spectrometry standardization between different samples, the artificial polypeptides also standardize sample preparation amongst different samples undergoing mass spectrometric analysis when using electrophoresis separation prior to mass spectrometric analysis. Methods of the present invention also include methods for designing artificial polypeptides with peak to peak continuous liquid chromatography elution profiles spanning the complete or partial analyte elution profile for organic and biological molecules. Also included are the artificial polypeptides predigested with protease, which is compatible for use in experiments with native PAGE, in-solution proteolytic digestion of polypeptides, and small organic molecules undergoing fractionation separation followed by mass spectrometric evaluation.

Description

FIELD OF THE INVENTION[0001]The present invention relates broadly to the field of mass spectrometry. The present invention relates more specifically to the quantification of analytes, specifically to mass spectrometric quantitative analysis of analytes (including proteins, peptides, small molecule chemicals, and other organic or inorganic compounds). The present invention further provides methods of designing and producing chemical and biological reporter sets that are useful in determining mass spectrometry detection efficiency.BACKGROUND OF THE INVENTION[0002]Mass spectrometry (MS) is an excellent technique for quantifying the amount of known and unknown substances in complex mixtures. Although, MS does have issues that can adversely affect the ability to assay or measure chemical and biological analytes. One problematic issue is ion suppression or enhancement in electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix assisted laser desorption io...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): G01N30/72H01J49/00
CPCH01J49/0027G01N30/7233G01N33/6848G01N2030/8831G01N30/96
Inventor HEAVEN, MICHAEL R
Owner UAB RES FOUND
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products