1. Principles of LC-MS Coupling
Analytical Biotechnology
Principles of LC-MS Coupling
Dr. Isam Khalaila Biotechnology Engineering Department

2. Mass Spectrometer as Detector
Sensitive
Picomoles of peptides;
Specific
High resolution
Accurate mass
Informative
Confirmation
Structure
Aston discovered that even stable atoms have isotopes in 1919 – Nobel prize followed.

3. MS FOR BILOGICAL SAMPLES
THE CRUCIAL ISSUES IN BIOLOGICAL SAMPLES ARE:
The diversity of proteins content
low abundant proteins (hard to detect)

4. Proteomics Challenge Complex samples require simplification
Intact proteins are too big
Large numbers of samples/replicates to be run
Automation needed

5. Proteomics is the Analysis of Complex Mixtures
Separation of proteins/peptides is a necessity
Intact protein separation
Centrifugation
Filtering LC
Isoelectric focusing
Gel electrophoresis
Peptide separation
Capillary electrophoresis

6. Liquid Chromatography
Coupling LC to MS can simplify samples and solve the problem of protein diversity and low abundant analytes.

7. LC-MS Application in Forensic Science
Analytes were separated at 40 °C on a Luna 3u PhenylHexyl column (50x2mm i.d., 3 μm)
Maralikova and Weinmann J. Mass Spec. 2004; 39: 526–531

8. LC-MS Application in Forensic Science MS/MS of THC

9. LC-ESI-MS Analysis of D9-tetrahydrocannabinol in oral fluid samples
LC–MS chromatogram of an oral fluid sample of a cannabis consumer. Peak at t = 3.4 min: 51 ng/mL D9-THC: (a) oral fluid extract (m/z ); (b) oral fluid extract (m/z ).
LC–MS analysis of blank oral fluid samples: (a) oral fluid extract (m/z ); (b) oral fluid extract (m/z ).

10. ESI mass spectrum of D9-THC?
H. Teixeira et al. Forensic Sci. Int :205–211

11. LC prinsiples: to refresh your memory
What is the purpose?
Analytical
Preparative
What are the molecular characteristics of the analyte and sample?

12. Chromatography Scales
Chromatography Objective
Analytical
Information ID and concentration
Semi Preparative
Small amount of purified compound < 0.5 gr
Preparative
Large amounts of purified compound > 0.5 gr
Industrial
Manufacturing quantities gr-kg

13. Analyte properties Charge Hydrophobicity Affinity
Positive/negative
Hydrophobicity
Affinity
“lock and key” sites
Solubility & stability
pH, ionic strength, organic solvents
Molecular weight

14. Analytical Requirements
Linearity
Precision
Accuracy
Sensitivity
Assay reproducibility
Robustness

15. Method Efficiency
Two of the Major Factors Involved in “Efficiency” are:
Number of Theoretical Plates
Resolution

16. Efficiency of HPLC results from:
Resolution
degree of separation between analyte and other species present in mixture
Band spreading
selectivity
Recovery
mass recovery
activity recovery
Capacity

17. Resolution of Separation

18. Resolution and Peak Separation

19. Number of Theoretical Plates (N)
H = Theoretical Plate Height
L = Length of the Column.
N = L / H

20. Particle Size and Flow Rate van Deemter Equation
H = A+ B/u + u [CM+CS]
Height Equivalent to Theoretical Plate
Linear Velocity
HETP U
Lowest HETP => Optimum Plate Count
{cm/sec}
HETP PLATES H
C Term
(kinetics of the analyte between mobile and stationary phase )
B Term
(Axial Diffusion)
Add the 3 terms to obtain final “van Deemter Curve”
A Term
(Particle size and how well bed was packed)

21. Optimization steps Select the mode pH map Optimize gradient/elution
gradient slope
eluent concentration
Loading study
overload: peak width and shape

22. Reverse phase (RPC)
Stationary phase hydrophobic and mobile phase hydrophilic
column: silica, polystyrene covalently modified with alkyl chain 3-18 C’s
EX: octadecylsilane (ODS) - C18
mobile phase: buffered water + organic solvent (propanol, CH3CN, CH3OH)
gradient elution

23. Ion-Exchange (IEC)
Ion exchange interactions between cationic or anionic analyte and stationary phase bearing opposite charge
stationary phase: polystyrene, silica modified with functional groups such as quaternary amines
mobile phase: buffer containing increasing concentration of salt (NaCl, MgCl2, K3PO4, NH4SO4)
gradient elution

24. Analyzed molecules Proteins Peptides Nucleic acids
Pharmaceutical products

25. What Factors Are Used in Selecting an Efficient Procedure?
Efficiency deal with: Cost, speed, ease, safety, accuracy, precision, sensitivity, and freedom from interference.
To achieve this, we must select an appropriate mobile phase, stationary phase (column), detector, and instrument variables (column temperature, flow rates, etc.).

26. Velocity and Stationary phase What is the Small Particle Advantage ?

27. Effects of linear velocity
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time (min) 20 40 60 80
100
120
140
160
180
2.99
1.24
3.92
2.45
4.82
5.65
1.92
0.78
2.51
1.58
3.09
3.60
1.20
0.49
1.57
0.98
1.93
2.25
Hypersil GOLD C18, 50 x 2.1 mm, 5 um
Mobile phases: 85% Water and 15%ACN
Pressure: 42 bar
Flow rate: 0.5 mL/min
Detector: 230 nm
Sample: 60 ng of six barbiturates
Pressure: 69 bar
Flow rate: 0.8 mL/min
Pressure: 22 bar
Flow rate: 0.3 mL/min

28. Particle size advantage
Hypersil GOLD C18, 50 x 2.1 mm, 5 um
Mobile phase: 15% ACN and 85% Water
Flow rate: 0.5 ml/min
Pressure: 42 bar = 609 psi
Detector: 230 nm
Sample: 60 ng
Hypersil GOLD C18, 50 x 2.1 mm, 1.9 um
Mobile phase: 15% ACN and 85% Water
Flow rate: 0.5 ml/min
Pressure: 275 bar = 3989 psi
Detector: 230 nm
Sample: 60 ng

29. Even Smaller Particle SizeAU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00
2.00
4.00
6.00
8.00
10.00
12.00
15.00
1.00
3.00
5.00
4.8 µm, 0.2 mL/min
354 psi
Ultra Pressure Liquid Chromatography
1.7 µm, 0.6 mL/min
7656 psi

30. Sensitivity Increased as Particle Size Decreased

31. Resolution and Analysis Time Change with Column Length
Where: Rs = Resolution L = Column length T = Analysis time
Column: 4.6 x 250 mm
Rs1 : 3.6 T1: 17 min.
Column: 4.6 x 75 mm

32. Small Particle Reduce Analysis Time While Maintaining High Resolution

33. Effect of Particle Size
Digest of complex protein mixture
5 µm Magic C18
3 µm Reprosil C18

34. Advantage of UPLC

35. Melamine in Food

36. Melamine quantitation and characterization=?
113
110

37. High throughput HPLC for proteomics approach
Low MW
High MW
acidic
basic

38. Making proteins to fit mass spec range…

39. Peptide LC Separation Simplification of mixtures
Removal of contaminants
Concentration effect
Direct coupling to mass spectrometer
Automation

40. LC-Electrospray Coupling

41. Miniaturisation of LC Columns
4.6 mm ID
1 mm ID
180 um ID
75 um ID
10 um ID
Sample load
Speed of analysis
Sensitivity

42. Details of Plumbing with ColumnhV
PicoFrit
LC Flow

43. Concentration effect Protein digest (peptides) Peptide total 100 fmol
Injection volume 2µL
Peptide conc. 50 fmol/µL
Flow rate 0.1 uL/min
Elution time 0.4 min
Peptide concentration
0.1 l/min * 0.4 min = 2500 fmol/ l
Concentration factor = 50 times 25 26 27 28 29 30 31 32 33
Time (min) 20 40 60 80
100
27.29
30.40
582.54
536.76
25.94
424.96
25 sec

44. Nano LC-MS (ESI or MALDI)
Bodnar et al. J Am Soc Mass Spectrom 2003, 14, 971–979

45. Ribosomal proteins identification using LC/ESI/MS/MS and LC/MALDI/MS/MS
Bodnar et al. J Am Soc Mass Spectrom 2003, 14, 971–979

46. Comparison of LC/ESI/MS and LC/MALDI/MS Spectra

47. Comparison of LC/ESI/MS/MS and LC/MALDI/MS/MS Spectra

48. LC/ESI/MS/MS and LC/MALDI/MS/MS Identified Bovine Ribosomal Proteins

49. Multidimensional Chromatography
Benefit: Sensitive
Drawback: Not Robust
Yates J.R. (1999) Nature Biotechnol. 17,
Yates J.R. (2001) Nature Biotechnol. 19,
Nägele et al. (2004) J. Biomol. Tech. 15:143.

50. Schematic flow of multi-dimensional LC-MS
Protein Complex
Ion-exchange-HPLC
Reverse=phase-HPLC
Tandem-MS
Database search
Peptide sequence
Identification of protein
Proteolytic degradation

51. 1 SCX column 2 RP column 2 micro-flow pumps 10-port valve
Example: Plumbing 2D LC
1 SCX column
2 RP column
2 micro-flow pumps
10-port valve

52. Plumbing Design ESI RP 2 SCX RP 1 WASTE AS Loop Water/ACN Water/Salt10 9
SCX 1 8 2 7
ESI
Water/ACN 3 6 4 5
RP 1
WASTE
Water/Salt

53. Sample Loading via Autosampler
AS Loop
RP 2 10 9
SCX 1 8 2
ESI 7
Water/ACN 3 6 4 5
RP 1
WASTE
Water/Salt

54. Elution - First Batch of Peptides
RP 2
SCX 10 9 1 8
Water/ACN 2 7
ESI 3 6 4 5
RP 1
WASTE
Water/Salt

55. Simultaneous Loading – Second Batch
RP 2
SCX 10 9 1 8
Water/ACN 2 7
ESI 3 6 4 5
RP 1
WASTE
Water/Salt

56. Simultaneous Loading – Third Batch
RP 2
SCX 10 9 1 8
ACN gradient 2 7
ESI 3 6 4 5
RP 1
WASTE
Salt step

57. LC Trace of 2D Separation
[NH4Cl] = 20 mM
40 mM
60 mM
80 mM
100 mM
150 mM

58. LC Trace of 2D Separation
200 mM
250 mM
300 mM
500 mM

59. Proteins Identified - Comparison
Sample 1D 2D
A431 Cells
341
864
Shieh et al ASMS 2002 Poster.

60. 1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome
Y. Guo et al. Proteomics 2005, 5, 4608–4624

61. 2D LC/MS Analysis of Membrane Proteins from Breast Cancer Cells
Schematic diagram of membrane and peptide preparation from MCF7 and BT474 cells.
Xiang et al. J. Proteome Res. 2004, 3:

62. 2D LC/MS Analysis of Membrane Proteins from Breast Cancer Cells
Av. Spectrum interval min.
RP18 BPC of 25mM amm formate SCX frac.
Xiang et al. J. Proteome Res. 2004, 3:

63. LC-MS Coupling: Benefits
Reduces complexity of mixtures
Removes chemical interferences like salts, detergents
Improves the ionisation of compounds
Increases sensitivity by concentrating the analyte
Easy to automated to enable high throughput

64. End