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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)
2
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)
• 2 derivatizing agents investigated
Benzoic acid, 4-amino-, ethyl ester (ABEE)
Benzoic acid, 4-amino-, 2-(diethylamino)ethyl ester (ABDEAE)
3
MS (+ ESI) maltohexaose
50 pmol (free sugar) 5 pmol (ABEE-derivatized)
100 fmol (ABDEAE-derivatized) 10 fmol (ABDEAE-derivatized)
MS/MS (+ ESI) maltohexaose-ABDEAE
100 pmol/uL 1 pmol/uL
4
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)
February 2009 JMSDonald H. Chace newborn screening
Inherited metabolic diseases
Phenylketonuria (PKU)Reduction in Phenylalanine to tyrosine
metabolism (leads to increase in Phe levels)
5
Derivatization to achieve selectivity :Newborn Screening for Disease
Blood sample from newborn collected onto dried filter paper
MS/MS is a fast, accurate, robust disease screening
method requires no chromatographic separation
extremely rapid: 2 min. per sample
methanol extraction of samples collected onto dried filter paper removes proteins and salts
Extracted sample derivatized to butyl esters
acidified butanol (3N HCl) or butanol with acetyl chloride
Product Ion Scan of Phenylalanine butyl ester shows loss of m/z 102
6
Phenylalanine, butyl ester
m/z 222
H3N CH C
CH2
O
O
Histidine, butyl ester
butyl ester amino acid derivatives lose 102 in MS/MS Examples:
ControlBlood sample fromNormal Newborn
Masses of deuterated internalstandards are underlined
Rerence: Chace, Chem. Rev. 2001, 101: 445-477
Neutral Loss Spectra from Newborn Screen
Phe
Tyr
Blood sample fromnewborn diagnosedwith phenylketonuria
Phe
Tyr
Phenylketonuria
7
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 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
8
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)
9
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?
10
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
cartridges
96 well plate
disk
http://solutions.3m.com/wps/portal/3M/en_US/Empore/extraction/
11
Solid-Phase Extraction (cont’d)
• Types of Chromatography
– Normal Phase
• Non-polar mobile phase
• Polar stationary phase
– Reversed Phase Most common
• Polar mobile phase
• Non-polar stationary phase
– Ion Exchange
• Buffer/Ionic mobile phase
• Cationic/Anionic exchange stationary phase
Manufacturer Brand Name
Waters SEP-PAKOASIS
Varian BondElute
Baker BakerBond
3M Empore
Supelco Supelclean
+ Many Others
Solid-Phase Extraction - common protocol
• Procedure
Sample
Prepare: Homogenize, suspend,
centrifuge, etc…
Load onto conditioned cartridge
Wash off weakly retained interferences
with weak solvent
Elute product with strong solvent
Analyze: HPLC, GC-MS, LC-MS/MS
12
pure analyte(control)
engine oil contaminatedparking lot oil
same as b) after organicmatter removal by SPE
(KNO3)nK+
Gapeev, A. and Yinon, J. J. Forensic Sci. 2004, 49
http://www.millipore.com/techpublications/tech1/tn072
13
-40000
-20000
0
20000
40000
Co
un
ts
1000 1500 2000 2500 3000 3500 4000 4500
Mass (m/z)
ZipTip C18 Prep in PBS/Urea/NaCl
Standard Prep in PBS/Urea/NaCl
labiomed.org/pdf/sample_cleanup.ppt
SPE (ZipTip) of protein digest using C18 bed
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
Cartriges, disks, filters, plates
Dialysis/UltrafiltrationMolecular weight/size
SlideAlyzer/tubing
Types of Separation Technologies for Molecules
14
Tubing or Slide A-LyzerOr Tube-O Dialyzer or 96 well plate formatDiff MWCO ranges0.1– 0.5 mL capacityUseful for biologicals
Sample
loading
here
Dialysis
Spin filters
polyethersulfone membrane
(Vivaspin, ex)
volumes from 100 μl to 20 ml,
with a range of molecular
weight cutoff values from Mr = 3 000 - 100 000
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
Cartriges, disks, filters, plates
Dialysis/UltrafiltrationMolecular weight/size
SlideAlyzer, tubing, spin filter
Distillation or Evaporation
Boiling point/vapor pressure
Destillator, He-purge
Precipitation Solubility
Types of Separation Technologies for Molecules
15
Very useful for messy/dirty protein samples
TCA*, Acetone, Ethanol precipitation methods
o Bring protein solution to 80% acetone using HPLC-grade acetone
o Incubate at –20oC overnight or in dry ice for 2-3 hours (don’t cut incubation time)
o Centrifuge 10 min at 4oC and carefully remove supernatant
o Wash pellet gently with two aliquots of 100% acetone at –20oC
o Dry sample briefly under vacuum and store sealed at –20oC
Precipitation
*TCA: trichloro acetic acid
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
Types of Separation Technologies for Molecules
Protein Mixture
1D SDS-PAGE
2D SDS-PAGE
pHMW
MW
Great clean-up tool (rid of salts, detergents, etc…)
Great concentration tool
Biological analytes
Various stains available – various detection limits
USE PRECAST GELS (polymer issue) if possible
Various size gels (spatial resolution)
Various MW ranges
Various pI ranges
PAGE: polyacrylamide gel electrophoresis
SDS: sodium dodecyl sulphate
16
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
HPLCGeneral molecule useProtein(s) Digest to peptides LC-MS/MS or MALDI-TOF-MS
Types of Separation Technologies for Molecules
Enzyme
Peptides m/z
Ab
un
danc
eMS
Protein
HPLC
Time (min)
UV
Abs
orba
nce
5 65
Abu
nd
ance
Abu
nd
ance
Abu
nd
ance
MS/MS
Most often for proteomics, chromatography clean-up is essential if not mandatory
(ie: LC-MS/MS)
Digestion is also
sample prepTrypsin (R/K)
Chymotrypsin (F/W/Y/L)
Pepsin (indiscriminate)
Others (CNBr, Formic Acid)
Separates based on combination of
hydrophobicity and molecular weight
17
http://www.lcpackings.com/
http://www.michrom.com
http://www.chem.agilent.comhttp://www.microlc.com/
http://www.eksigent.com/
http://www.waters.com
HPLC - hardware (often nano)
HPLC Column Configurations and Applications
Column Type
ID (mm) Length (mm)
Particle Size (m)
Flow Rate Ranges
Applications Sensitivity Increase
Nano 0.1-0.075 150 3.5 100-600 nL/min
Proteomics, Sample Limited PTM Characterization
2000-3700
Capillary 0.3, 0.5 35-250 3.5, 5 1-10 L/min
Peptide Mapping LC/MS
100
Micro Bore 1.0 30-150 3.5, 5 30-60 L/min
High Sensitivity LC/MS
20
Narrow Bore
2.1 15-150 3.5, 5 0.1-0.3 mL/min
Sample Limited. LC/MS
5
Analytical 4.6 15-250 3.5, 5 1-4 mL/min
Analytica; 1
Semi-prep 9.4 50-250 5 4-10 mL/min
Small Scale protein purification
--
Preparative 21.2 50-250 5, 7 20-60 mL/min
CombiChem purification
--
18
Enzyme HPLC MS/MS
Abun
danc
eAb
unda
nce
Abun
danc
e
m/z
Protein Peptides
HOW IMPORTANT IS REPRODUCIBLE and HIGH
QUALITY HPLC SEPARATION OF PEPTIDES ?
IT ALL DEPENDS ON GOALS/NEEDS/RESOURCES
0 10 20 30 40 50 60 70 80 90 100 110
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
707
34.22
713
34.51
737
35.73
555
26.96 773
37.47
561
27.22781
37.88
549
26.68787
38.17657
31.75503
24.53809
39.24457
22.3511
0.48 927
45.2421
0.95 395
19.39371
18.25
937
45.7549
2.26 1031
50.751201
60.14141
6.881321
66.75189
9.24 1951
105.001185
59.331815
96.63
1343
67.96
1589
83.09
1679
88.441509
78.212091
113.80
0 10 20 30 40 50 60 70 80 90 100 110
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
877
44.85
819
41.85
1005
51.55549
28.151207
62.22
669
34.19
999
51.23
76539.07 907
46.41561
28.771009
51.76911
46.62
543
27.86615
31.431017
52.18507
26.021295
66.941145
58.94
1067
54.83499
25.59
489
25.08399
20.67
349
18.20303
15.871309
67.66139
7.2213
0.57 1357
70.541441
76.14
1523
81.61
1653
90.461687
92.75
1817
101.40
1861
104.36
1945
109.90
2069
118.18
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
2345
73.52
201
6.22169
5.241633
51.911449
46.33
1769
56.06
217
6.70
2049
64.75
1937
61.34 2509
78.31
1417
45.35
2937
90.801485
47.43
1385
44.383321
101.912065
65.29
2181
68.692841
87.97269
8.323265
100.252621
81.603121
96.10493
15.463345
102.60369
11.49637
20.161077
34.451153
36.90
873
27.87
717
22.77
Nice chromatography
Decent chromatography
Poor chromatography
19
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
20
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
21
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
Gel Filtration Molecular Weight HPLC
Ion ExchangeCation or Anion affinity
FPLC
Affinity Chromatography DNA,RNA, Anti-body, peptides etc
HPLC
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
22
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
23
Sample Clean-up/Prep Conclusions (for proteomic applications)
•There are many different ways to get from protein/peptide to tandem mass spec
•What you use depends on what you are trying to find out e.g. identification, structural characterization, quantitation
•Choosing the best tool for the job can be very difficult and may require a combination of approaches
http://www.gbiosciences.com/
24
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/service-and-support/handbooks
Suggested Reading ListProtein ID from Gels
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.
25
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
26
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