1 Derivatization and Sample Prep for (Small) Molecules Árpád Somogyi CCIC MSP OSU Summer Workshop Why Derivatize? • To increase abundance of MH + for better MS/MS – Improve sensitivity for detection of oligosaccharides • To achieve selectivity in a detection scheme – Newborn screening for disease: Detection of amino acid imbalance, a marker of inherited disease • To distinguish an analyte from an interference – Analysis of methylmalonic acid in plasma & urine (differentiated from succinic acid – same mass)
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Derivatization and Sample Prep for (Small) Molecules
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Derivatization and Sample Prep for (Small) Molecules
Árpád Somogyi
CCIC MSP
OSU Summer Workshop
Why Derivatize?
• To increase abundance of MH+ for better MS/MS– Improve sensitivity for detection of oligosaccharides
• To achieve selectivity in a detection scheme– Newborn screening for disease: Detection of amino acid
imbalance, a marker of inherited disease
• To distinguish an analyte from an interference– Analysis of methylmalonic acid in plasma & urine (differentiated
from succinic acid – same mass)
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Why Derivatize?
• To increase abundance of MH+ for better MS/MS– Improve sensitivity for detection of oligosaccharides
• To achieve selectivity in a detection scheme– Newborn screening for disease: Detection of amino acid
imbalance, a marker of inherited disease
• To distinguish an analyte from an interference– Analysis of methylmalonic acid in plasma & urine (differentiated
from Succinic Acid)
Derivatization to Improve Sensitivity for Detection of Oligosaccharides
Reference: Yoshino et al, 1995, 76:4028-4031
• Poor ionization efficiency of free carbohydrate chains in ESI limits the utility of ESI-MS in structural carbohydrate studies
• Ionization efficiency enhanced in positive-mode ESI-MS (improved 5000-fold)
• To increase abundance of MH+ for better MS/MS– Improve sensitivity for detection of oligosaccharides
• To achieve selectivity in a detection scheme– Newborn screening for disease: Detection of amino acid
imbalance, a marker of inherited disease
• To distinguish an analyte from an interference– Analysis of methylmalonic acid in plasma & urine (differentiated
from succinic acid)
Derivatization to Distinguish an Analyte from an Impurity: Methylmalonic Acid (MMA) in Plasma &
Urine (Differentiated from Succinic Acid)
• Increased MMA is a specific diagnostic marker for propionate metabolism and acquired vitamin B12 deficiency
• Must overcome interference from the isomer succinic acid
MMA, MW = 118.09succinic acid, MW = 118.09plasma
(umol/L)
urine (mmol/mol creatine)
MMA 0-0.4 0-3.6Succinic acid 0-32 0.5-16
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Methylmalonic Acid Detected in Plasma & Urine (Differentiated from Succinic Acid)
• HPLC-MS/MS methods have been developed (QQQ) -MRMs
– replace GC-MS methods in a high-throughput environment
• extracted sample (SPE) derivatized to butyl esters
– extracted from plasma or urine, eluted and derivatized with HCl in n-butanol
• the method is demonstrated in this example using standards
• results of plasma & urine samples are in agreement with the same samples analyzed by the standard method (GC/MS)
References: Magera et al, Clinical Chem. 2000, 46 (11): 1804-1810
Schmedes et al, Clinical Chem. 2006, 52(4): 754-757
MS/MS of Derivatized MMA and Succinic Acid
MMA butyl ester
succinic acid butyl ester
Loss of C4H8
butyl groups(-112)
Loss of C4H8
butyl groups+ loss of H2O
(-130)
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MRM Extracted Ion Chromatograms Selected transitions m/z = 231/119 and 234/122 (MMA-d3)
1 = succinic acid 2 = MMA-d3 3 = MMA
1 nmol MMA
1000 nmol succinic acid
10 nmol MMA
10 nmol MMA
10 nmol MMA
100 nmol succinic acid
10 nmol succinic acid
1 nmol succinic acid
• To separate interfering species from analyte– Example: analysis of drug and metabolites in plasma need to
remove protein interferences
– Off-line or in-line from MS/MS detection
• To concentrate analyte– Example: Pesticides in drinking water
• Basic principle of sample clean up involves preferential binding of analyte over interfering species or vice versa, followed by elution to MS/MS
Separation technologies
essential in sample prep
Why Clean up samples?
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MethodSeparation based
onSeparation done
usingFurther steps
Liquid-liquid Extraction
Partitioning in one of two liquid phases
Glass ware
Types of Separation Technologies for Molecules
An immiscible solventis added to the sample which then separates into 2distinct liquid phases. Some sample analytes will go into the bottom phase (Aqueous), some will separate into the top phase (Organic)
“Trizol” – a form of liquid-liquidpartitioning of RNA, DNA and protein
“guanidinium thiocynate-phenol-chloroform”
• Large solvent consumption• Time/labor intensive• May need evaporation step• >1 extraction if mixture of analytes• Emulsions and contamination issues
Chomczynski P, Sacchi N. Anal Biochem. 1987 Apr;162(1):156-9
MethodSeparation based
onSeparation done
usingFurther steps
Liquid-liquid Extraction
Partitioning in one of two liquid phases
Glass ware
Solid-phase Extraction
Adsorption/ partitioning onto solid sorbent
Cartridges, disks, filters, plates
Types of Separation Technologies for Molecules
• Uses chromatographic particles• Packed-bed column cartridges or similar• Well established commercial technology
(1978)• 1000s literature refs• Clean extracts• Good recovery for polar analytes• Sample must be in liquid state• Driving force: gravity, pressure, vacuum• Automation
For protein identification using LC-MS/MS, chromatography
may not be an issue because one can rely on mass spectrum
resolving power of co-eluting peptides
For biomarker discovery, post translational modification (PTM) characterization and label free peptide quantitation, reproducible chromatography is very important
Contaminants To Avoid for LC-MS/MS Applications
• Ideal Salt and buffer concentrations are < 10 mM, there are various ways to clean-up the samples
• Desalting very important, especially with glycoproteins, oligonucleotides, and higher mass proteins– i.e. - less peak broadening, less overall interference, less
interference with matrix crystal formulation (MALDI MS applications)
• Preferred Solvents are H2O and ACN, avoid DMSO, DMF and other large polar solvents
• Storing samples in glass vials (Na & K contamination)– Store samples in Sarstedt vials only (minimizes polymer
contamination)
• Detergents, all types (a big no)• Protease inhibitors (remove these before sample
submission)• Glycerol Keller et al, Interferences and contaminants encountered
in modern mass spectrometry. Analytica Acta 2008, 627, 71-81
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Appendix Material
• Keller et al, Interferences and contaminants encountered in modern mass spectrometry. Analytica Acta 2008, 627, 71-81
MethodSeparation based
onSeparation done
usingFurther steps
Gel Electrophoresis (1D)
Molecular massGel (which acts like a molecular sieve) and potential
In-gel digestion of proteins to peptidesLC-MS/MS or MALDI-TOF-MSGel Electrophoresis
(2D)
Isoelectric point (pI; IEF) & Molecular mass
Gel, potential and ampholytes
Reverse Phase (C18, C8 or C4) chromatography
Combination of hydrophobicity and molecular weight
HPLC
Protein(s) Digest to peptides LC-MS/MS or MALDI-TOF-MS
Gel Filtration Molecular Weight HPLC
Ion ExchangeCation or Anion affinity
FPLC
Affinity Chromatography “pull down”
DNA,RNA, Anti-body, peptides etc
HPLC
Types of Separation Technologies for Molecules
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1
proteolysis
immuno -precipitate
LC-MS-MS
data miningalgorithmsIdentification
of components
Protein complex
anti -anti -
MS-MS spectra
1SDS-PAGE
excise bandsdigest
MALDI -TOF MSor LC-MS/MS
data miningalgorithms
Identificationof components
peptide mixture
Affinity Chromatography
MethodSeparation based
onSeparation done
usingFurther steps
Gel Electrophoresis (1D)
Molecular massGel (which acts like a molecular sieve) and potential
In-gel digestion of proteins to peptidesLC-MS/MS or MALDI-TOF-MSGel Electrophoresis
(2D)
Isoelectric point (pI; IEF) & Molecular mass
Gel, potential and ampholytes
Reverse Phase (C8 or C4) chromatography
Combination of hydrophobicity and molecular weight
HPLC
Protein(s) Digest to peptides LC-MS/MS or MALDI-TOF-MS
MudPIT (Multi-dimensional Protein Identification Technology
Cation Exchange & hydrophobicity (used for peptides; not for proteins)
HPLCOnline MS/MS analysis
Types of Separation Technologies for Molecules
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SCX RP
1.8 kV
HPLC
Multidimensional Protein Identification Technology (MudPIT)
Load peptide mixture
To MS
Salt Bump
RPSCX
To MS
RPSCX
To MS
RPSCX
Reverse Phase Gradient
MudPIT In chromatographic theory, theoretical plates of orthogonal separation columns back-to-back are multiplied rather than summed – that’s why it works
Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. . Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. (1996)Nature 379, 466–469.
2D gel review Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients. (2000) Electrophoresis. 21(6):1037-53.
Comparative 2D gels
B.Cooper , D. Eckert, N.L. Andon, J. R. Yates III and P. A. Haynes: “Investigative Proteomics: identification of an unknown plant virus from infected plants using mass spectrometry” – (2003), J Am. Soc. Mass Spectrom., Vol 14, no. 7, 736-741.
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MudpitLink A.J, Eng J, Schieltz D.M, Carmack E, Mize G.J, Morris D.R, Garvik B.M, Yates J.R, Direct analysis of protein complexes using mass spectrometry. (1999) Nature Biotechnology, 17, 676-682.Washburn MP, Wolters D, Yates JR III Large-scale analysis of the yeastproteome by multidimensional protein identification technology (2001) NatureBiotech. 19:242-247.
TAP Tagging
Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration. (1999) Nat Biotechnol. Oct; 17(10): 1030-2.Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. (2002) Nature 415, 141-147.Ho, Y., Gruhler, A., Heilbut, A., Bader, G. D., Moore, L., Adams, S. L., Millar, et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. (2002) Nature 415, 180-183.
Protein Modification MappingMacCoss M.J., McDonald W.H., Saraf A., Sadygov R., Clark J.M., Tasto J.J., Gould K.L., Wolters D., Washburn M., Weiss A., Clark J.I., Yates J.R. III. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc Natl Acad Sci USA. 2002 99(12):7900-7905.
DIGEUnlu M, Morgan ME, Minden JS (1997). Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis18:2071-2077.Somiari RI, Sullivan A, Russell S, Somiari S, Hu H, Jordan R, George A, Katenhusen R, Buchowiecka A, Arciero C, Brzeski H, Hooke J, Shriver C. (2003) High-throughput proteomic analysis of human infiltrating ductal carcinoma of the breast. Proteomics. 3:1863-73
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Carbohydrate MS/MS
Lattova E, Snovida, S., Perreault, H, and Krokhin O. Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry
J Am Soc Mass Spectrom. 2005 May;16(5):683-96.
Mass spectrometry of oligosaccharides. Zaia J., Mass Spectrom Rev. 2004 23(3):161-227.
Structural characterization of NETNES, a novel glycoconjugate in Trypanosoma cruzi epimastigotes. Macrae JI, Acosta-Serrano A, Morrice NA, Mehlert A, Ferguson MAJ, J Biol Chem. 2005 Apr 1;280(13):12201-11. Epub 2005 Jan 13.
Lipid and glycolipid MS/MS
Tong Y, Arking D, Ye S, Reinhold B, Reinhold V, Stein DC. Neisseria gonorrhoeae strain PID2 simultaneously expresses six chemically related lipooligosaccharide structures. Glycobiology. 2002 Sep;12(9):523-33.
Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate, Mahapatra S, Yagi T, Belisle JT, Espinosa BJ, Hill PJ, McNeil MR, Brennan PJ, Crick DC. J Bacteriol. 2005 187(8):2747-57