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

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.

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

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

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

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

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

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

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 315.31); (b) oral fluid extract (m/z 193.13). LC–MS analysis of blank oral fluid samples: (a) oral fluid extract (m/z 315.31); (b) oral fluid extract (m/z 318.00).

ESI mass spectrum of D9-THC ? H. Teixeira et al. Forensic Sci. Int. 2005. 150:205–211

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

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

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

Analytical Requirements Linearity Precision Accuracy Sensitivity Assay reproducibility Robustness

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

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

Resolution of Separation

Resolution and Peak Separation

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

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)

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

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

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

Analyzed molecules Proteins Peptides Nucleic acids Pharmaceutical products

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.).

Velocity and Stationary phase What is the Small Particle Advantage ?

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

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: UV @ 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: UV @ 230 nm Sample: 60 ng

Even Smaller Particle Size AU 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

Sensitivity Increased as Particle Size Decreased

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

Small Particle Reduce Analysis Time While Maintaining High Resolution

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

Advantage of UPLC

Melamine in Food

Melamine quantitation and characterization =? 113 110

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

Making proteins to fit mass spec range…

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

LC-Electrospray Coupling

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

Details of Plumbing with Column hV PicoFrit LC Flow

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

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

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

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

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

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

Multidimensional Chromatography Benefit: Sensitive Drawback: Not Robust Yates J.R. (1999) Nature Biotechnol. 17, 676-682. Yates J.R. (2001) Nature Biotechnol. 19, 242-247. Nägele et al. (2004) J. Biomol. Tech. 15:143.

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

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

Plumbing Design ESI RP 2 SCX RP 1 WASTE AS Loop Water/ACN Water/Salt 10 9 SCX 1 8 2 7 ESI Water/ACN 3 6 4 5 RP 1 WASTE Water/Salt

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

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

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

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

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

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

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

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

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, 3:1278-1283

2D LC/MS Analysis of Membrane Proteins from Breast Cancer Cells Av. Spectrum interval 64-65 min. RP18 BPC of 25mM amm formate SCX frac. Xiang et al. J. Proteome Res. 2004, 3:1278-1283

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

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