Nov 19 Lecture 7: 1.Advanced Separations Methods: HPLC · PDF fileLecture 7: 1.Advanced...

Lecture 7:

1.Advanced Separations Methods: HPLC vs. UPLC

vs. HILIC

2. Nanoflow vs. ESI

3. Applications;

4.Laser capture miscrodissection (LCM)

1 ME 330.80: Role of Chromatography & Mass

Spectrometry in Biological Research

http://www.hopkinsmedicine.org/mams/

Adriana Bora, PhD

Nov 19th 2012




Technologies for Proteomics

Nature biotechnology 28, 695-709, (2010)




“omics” Workflow Tissue Blood CSF Plasma Urine Serum Sample

Sample preparation

Peptide/Protein Extraction, Desalting, Abundant Protein Depletion, Detergent Removal, etc.

Separation HPLC/UPLC, HILIC, SEC, IEC, etc.

Mass Spectrometry

LC-MALDI, MALDI-TOF, QTOF, IT, Orbitrap, etc.

MASCOT, SEQUEST, PROTEIN DISCOVERER, etc. Data Analysis

Liquid Chromatography

• Defined as separation of components of a mixture based upon the rates at which they elute from a stationary phase typically over a mobile phase gradient.




• Ion exchange chromatography • Size exclusion chromatography • Adsorption chromatography

Different Phases

• Normal Phase – This is where the stationary bed is strongly polar (silica gel) and the mobile phase is largely non-polar such as hexane.

• Reverse Phase – The stationary phase is non-polar and the mobile phase are polar liquids such as methanol, acetonitrile, or water. The more non-polar substances have longer retention.

Reference 1 5

ME 330.80: Role of Chromatography & Mass



Elution Types

• Isocratic – where the eluent is at a fixed concentration.

• Gradient – where the eluent concentration and strength are changing.

Reference 1 6




Types of Liquid Chromatography

Gravity Chrom. Tsvett, 1903

Flash Chrom. 1978

HPLC 1952 UPLC 2004 (TLC) Paper Chrom.




HPLC Characteristics • Columns have small internal diameters (2-10 mm)

usually made with a reusable material like stainless steel

• High inlet pressures of several thousand psi’s and controlled flow of mobile phase

• Precise sample introduction and small sample requirements

• Special continual flow detectors that use small flow rates and low detection limits

• Some are equipped with automated sampling devices

• Rapid analysis with high resolution

Reference 3 8




Stationary Phase in HPLC

• Particle size 3 to 10 µm packed tightly with a pore size of 70 to 300 Å

• Surface area of 50 to 250 m2/g

• Bond phase density – number of adsorption sites per surface unit (1 to 5 per 1 nm).

• Typical surface coatings:

Normal phase (-Si-OH, -NH2)

Reverse phase (C8, C18, Phenyl)

Anion exchange (-NH4+)

Cation exchange (-COO-)

Reference 3 9




Mobile Phase in HPLC

• Purity of the solvents

• Detector compatibility

• Solubility of the sample

• Low viscosity

• Chemical inertness

• Reasonable price

Reference 3 10




Path of Mobile Phase

Mobile Phase degassing

Mobile Phase reservoir

Mobile Phase mixing

HPLC Pump Rotary Sample Loop injector

HPLC Column

HPLC Detector







How does it work HPLC/UPLC Technology?

Phytochem. Analysis, 2010, 21, 33-47, Allwood &Goodacre

Pumps

A B Line wash

Syringe wash

Solvent proportioning valve

Flow valve and monitor

Autosampler injector

HPLC/UPLC column

Waste

Detector

Secondary Detector: UV/Vis MS NMR

Chromatogram

Solvent A 5% Aq, ACN +0.1% FA Solvent B 95% Aq, ACN +0.1% FA

HPLC Columns

• HPLC Columns come in various sizes and many factors involving your analyte or the function of the column should be considered when selecting the appropriate one.

• Some common dimensions: 10, 15, and 25 cm in length;

• 3, 5, or 10 mm diameters;

• 4 to 4.6um internal diameters

Reference 3 13




HPLC Detectors

• Most HPLC instruments are equipped with optical detectors.

• Light passes through a transparent low volume “flow cell” where the variation in light by UV Absorption, fluorescent emission, or change in refractive index are monitored and integrated to display Retention Time and Peak Area.

• Typical flow rates are 1 mL/min. and a flow cell volume of 5-50 µL.

Reference 3 14




Common HPLC Detectors

• Refractive Index (RI) - universal

• Evaporative Light Scattering Detector (ELSD) – universal

• UV/VIS light – selective

• Fluorescence – selective

• Electrochemical (ECD) selective

• Mass Spec (MS) - universal

Reference 3 15




Mass Spectrometer

• Thermospray – mobile phase is directed to a capillary column that is heated and points at a skimmer cone. (Too much build up on orifice)

• Electrospray (ESI) – analytes are charged upon exiting the capillary tube and cross sprayed with nitrogen. The charge particles cause a “Coulomb explosion” making smaller droplets of analyte to enter the skimmer cone.

• Atmospheric Pressure Chemical Ionization (APCI) – Analyte is heated by a ceramic tip on the column, cross flow of nitrogen decreases the droplet size, and a “corona discharge” charges the particles to enter the detector.

Reference 3 16




Why HPLC?

• HPLC works with compounds of higher molecular weights and polarity.

• Many biological samples are charged such as DNA and proteins.

• HPLC can be used with larger sample sizes and sample recovery to continue synthesis

• Good at separating stereoisomers; techniques that employ heat (GC) can cause racemization during analysis.

Reference 3 17




UltraPerformance Liquid Chromatography

(UPLC ) Technology • In 2004, further advances in instrumentation and

column technology were made to achieve very significant increase in:

RESOLUTION

SPEED

SENSITIVITY

Increase separation EFFICIENCY

• Columns with smaller particles [<1.7um]

• Mobile phase delivery is done at >15,000psi




Contrasting HPLC and UPLC

• UPLC gives faster results with better resolution

• UPLC uses less of valuable solvents like acetonitrile which lowers cost

• The reduction of solvent use is more environmentally friendly

Reference 6 19




UPLC columns

an ethylene bridged hybrid (BEH) structure • Superior mechanical strength • Efficiency • High pH stability and peak shape for

bases • C8; C18;Phenyl; HILIC • pH range 1-12 • Max pressure 15,000psi • Particle size 1.7um • Pore diameter/volume 130A 0.7 mL/g • Surface Area 185 m^2/g

• Peptides • Proteins • Oligonucleotides DNA/RNA • Amino acids




UPLC principle

The evolution of particle sizes over the last three decades.

Pla

te h

eig

ht

Why is UPLC more efficient

• Peak capacity (P) is the number of peaks that can be resolved in a specific amount of time.

• P is proportional to the inverse of the square root of the Number of theoretical plates (N): N = L/H

• Lower plate heights generate a smaller number of plates

• Plate heights are correlated through the Van Deemter equation







Why we need UPLC Technology?

• Metabolomics is the comprehensive assessment of endogenous metabolites of low-molecular weight (<1,000 Da)of a biological system.

• These small molecules, including peptides , amino acids , nucleic acids , carbohydrates , organic acids, vitamins , polyphenols , alkaloids and inorganic species act as small-molecule biomarkers that represent the functional phenotype in a cell , tissue or organism.

• Applications: drug discovery, toxicology, nutrition, cancer, natural product discovery, etc.

• These large-scale analyses of metabolites are intimately bound to advancements in ultra-performance liquid chromatography–electrospray (UPLC) technologies and have emerged in parallel with the development of novel mass analyzers and hyphenated techniques.




Chromatograms of simvastatin

24

• hypolipidemic drug • control elevated cholesterol

Chromatogram showing separation of Telmisartan and its degradation products in a mixture of stressed samples a) UPLC

b) HPLC

Angiotensin II receptor antagonist used to control hypertension

Reference 3 26




Reference 3 27




Hydrophilic Interaction Liquid Chromatography (HILIC)

• The principle of HILIC was described by Samuelson and Sjostrom (1952) for the separation of monosaccharides using anion exchange rasin as stationary phase; Samuelson O, Sjöström E. 1952. Utilization of Ion Exchangers in Analytical Chemistry. XXIV. Isolation of Monosaccharides. Sven Kem Tidskr64: 305–314.

Cubbon et al, Jun 2009, Mass Spec. Reviews; Metabolomic applications of HILIC–LC–MS

• The mechanism of retention is based on the hydrophilic partitioning of the analytes into the water-enriched stationary phase, and weak electrostatic interactions with either the positive or negative charge of the functional group. • HILIC separates polar molecules.

• A sulfoalkylbetaine zwitterionic stationary phase.

• Like the SCX method mentioned above, HILIC chromatography can be used to enrich

PTM-modified sub-populations from complex biological samples.

• Specifically, HILIC has been shown to enrich for glycosylation, N-acetylation, and phosphorylation. Peptides with N-acetyl modifications will elute as an early sub-fraction during a HILIC separation at relatively low water/organic mobile phase conditions. Phosphopeptides will elute within the middle of a HILIC separation, but with little contamination from non-phosphorylated species. Glycosylated peptides will be retained on the HILIC column while most sample components elute off; once high water/organic mobile phase ratios are reached, the remaining glycosylated peptides will elute as a purified sub-proteome.

• HILIC offer a good alternative to RP chromatography for the analysis of highly polar metabolites such as carbohydrates, their phosphorylated derivatives, and glycolytic intermediates, which are poorly retained on RPs.

Reference 3 28




HILIC application

Reference 3 29




HILIC applications

In real life we mostly use orthogonal separations

Gauci et al, J. Proteome Res. , 2009, 8(7), pp 3451-3463

2000

Nucleic proteins

• NanoLC (nLC) is named after the low flow rate (200-300 nL/min).

• This uses very low sample volumes and (1µL) very high selectivity and sensitivity are possible.

• nLC-ESI-IT-MS/MS is mostly used for the identification of proteins from very complex mixtures.




NanoLC- ESI –MS

32 32

Membrane Trap

SDS PAGE

Column

Gel Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE)

J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573

+ -

Anode

Chamber

Cathode

Chamber

Collection

Chamber

Courtesy of John Tran

32 32 ME 330.80: Role of Chromatography & Mass



+ Anode – Cathode

Membrane Trap

SDS-PAGE Column

Collection Chamber

Top Down Proteomics

Front-end separation

Mass spectral data acquisition

Protein identification by database search

Intact protein separation based on molecular weight:

Gel Eluted Liquid Fraction Entrapment Electrophoresis

(GELFrEE)

Tran, J. C.; Doucette, A. A., Anal. Chem. 2008, 1568-1573

Time (60-90 min)

MW

Courtesy of Neil Kelleher

33 33 33 33




34 34

Tran, J.C.; Doucette, A.A., Anal. Chem. 2008, 80, 1568-1573

Gel Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE)





35

15 15.5 16 17 18 20 22 24 26 28 30 35 40 45 60 75 90 std

collection time (min)

25 27 30 33 36 42 48 54 60 66 72 87 102 112 157 202 std

212 100

54

39

30

20

7.3

212

100

54

39

30

20

7.3


Mol W

t (k

Da)

Mol W

t (k

Da)


1 cm Column

3 cm Column

GELFrEE Separation of Bacterial Proteome

•1cm column gives faster and better separation over a broad mass range




36 36


GELFrEE Recoveries

BSA

Cytochrome C

Ubiquitin

40 ng Loading




37 37

gel

columns

collection

chamber

gasket gasket

cathode

chamber

anode

chamber

plate

bolts

dialysis

membrane

plate

plate

plate - +

power terminals

Multiplex GELFrEE


Load Collect





38

Load Collect

Multiplex GELFrEE




39

Increased Loading Capacity

250 150 100

75 50 37

25

15

10

kDa

250 150 100

75 50 37

25

15

10

kDa

20


16 17 18 19 20 21 22 24 26 28 30 35 40 45 60 75 90 std

16 17 18 19 20 22 24 26 28 30 35 40 45 60 75 90 std

Overloaded Column

Pooled Fractions from Equivalent Overloaded Amount

800 µg /column

800µg loaded in 8 different channels :100 mg / column

J.C

. T

ran

; A

.A. D

ou

ce

tte

, A

na

l. C

he

m. 2

00

9, 8

1, 6

20

1-6

209

0.4mm id

0.4mm id




40

Increased Throughput Fractions 1-16


16 17 18 19 20 22 24 26 28 30 35 40 45 60 75 90 std

250 150 100

75 50 37

25 20

15

10

kDa

250 150 100

75 50 37 25

15

10

250 150 100 75 50 37 25 20

15

10

kDa

kDa

Yeast

Bacteria

Urine





41

High reproducibility of different in channels


800ug per column

100ug per column

S.cerevisiae Proteome




42

•1120 proteins •428 unique proteins •1% false positive rate

S.cerevisiae Proteome

Identified proteins with LC MSMS




Adriana Bora; Carol Anderson; Muznabanu Bachani; Avindra Nath; Robert J. Cotter; J. Proteome Res. 2012, 11, 3143-3149.




• Analyzing CSF using GelFrEE –LC-MS/MS

Bottom Up Proteomics

400




Unseparated human CSF proteome

Adriana Bora; Carol Anderson; Muznabanu Bachani; Avindra Nath; Robert J. Cotter; J. Proteome Res. 2012, 11, 3143-3149.

A) Silver stained images showing GelfrEE fractions for visualization of the protein

separation found in each fraction.

B) and C) show the reproducibility of the separation technique.

Separated human CSF proteome with GelFrEE

Sample Preparation

Affinity Column

4 min Tryptic

Digestion

On-Column

Desalting

C18 Reverse Phase HPLC

SRM-MS Detection




• Reduces variability to under 10% • Cutting sample prep time down to 10min • Reducing operating cost by 50%

Perfinity – Shimadzu 8030 triple quadrupole MS




14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 min

-150000

-100000

-50000

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

uV

1min

2min

4min

6min

8min

Insulin protein digested on trypsin column using

Perfinity System

Undigested protein




0

10

20

30

40

50

60

70

80

90

%

16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0 31.0 min

15

20

25

30

35

40

45

50

55

60

65

70

mAU

CSF 30min gradient- 6min digestion-60 %B -40C

Separation of CSF proteins using the Perfinity

System




Laser capture miscrodissection (LCM)




Applications of LCM




Cont…







Cont….

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