derivatization and sample prep for (small) molecules

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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Why Derivatize?





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)

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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?





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)

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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

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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

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Why Derivatize?





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




• 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


onSeparation done

usingFurther steps



Glass ware

Solid-phase Extraction

Adsorption/ partitioning onto solid sorbent

Cartridges, disks, filters, plates


• 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/

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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

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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

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-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


onSeparation done

usingFurther steps



Glass ware



Cartriges, disks, filters, plates

Dialysis/UltrafiltrationMolecular weight/size

SlideAlyzer/tubing


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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


onSeparation done

usingFurther steps



Glass ware



Cartriges, disks, filters, plates

Dialysis/UltrafiltrationMolecular weight/size

SlideAlyzer, tubing, spin filter

Distillation or Evaporation

Boiling point/vapor pressure

Destillator, He-purge

Precipitation Solubility


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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


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


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

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onSeparation done

usingFurther steps




(2D)



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


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

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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

--

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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

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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


onSeparation done

usingFurther steps




(2D)



Reverse Phase (C18, C8 or C4) chromatography


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


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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


onSeparation done

usingFurther steps




(2D)



Reverse Phase (C8 or C4) chromatography


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


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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

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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/

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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.

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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

derivatization and sample prep for (small) molecules

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