Condensed phase ionisation techniques: spray methods Lecture 5 Condensed phase ionisation techniques: spray methods

At the end of this lecture you should be able to: describe the ion formation models in ESI-MS calculate molecular weights and charge states from low- and high-resolution ESI-MS spectra

Ionisation Techniques: Overview Gas-Phase Methods Electron Impact (EI) Chemical Ionization (CI) Desorption Methods Secondary Ion MS (SIMS) and Liquid SIMS Fast Atom Bombardment (FAB) Laser Desorption/Ionization (LDI) Matrix-Assisted Laser Desorption/Ionization (MALDI) Spray Methods Atmospheric Pressure Chemical Ionization (APCI) Electrospray (ESI)

Condensed phase ionisation techniques (2): spray methods Overview Thermospray APCI: Atmospheric pressure chemical ionisation APPI: Atmospheric pressure photoionisation Electrospray ionisation

Atmospheric pressure chemical ionisation (APCI) For non-polar and thermally stable compounds < 1500 Da Useful to combine with liquid chromatography Spray needle/ Capillary Ions Spray To mass spectrometer Flow Cone Nebuliser gas Skimmers Corona discharge Vacuum Atmospheric pressure www.chm.bris.ac.uk/ms/theory/apci-ionisation.html

Electrospray Ionisation (ESI) Nobel Prize to Fenn in 2002 Also atmospheric pressure ionisation Very versatile Also works for (very) large (bio)molecules, including proteins, nucleic acids, carbohydrates Softest ionisation technique of all Spray needle/Capillary Spray To mass spectrometer Flow 3-4 keV 2-10 ml/min Cone Skimmers Atmospheric pressure Vacuum http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html

Electrospray Ionisation (ESI) Flowrates: 2 to 10 ml/min: Best interface for LC/MS Can be combined with almost any mass analyser Common: TOF, Ion Trap, Quadrupole, FT-ICR Uses: Mass detection, structure elucidation, protein folding, H/D exchange, protein sequencing….

Sample characteristics Common solvents: mixtures of water with acetonitrile or methanol Usually with added acid (acetic, formic), < 1% Can’t tolerate (non-volatile) salt or buffers Can do positive or negative electrospray: selected by capillary voltage

What it looks like

Ionisation models Ion evaporation model Spray needle tip Multiply charged droplet Ion evaporation model Spray generates multiply charged droplets Solvent evaporation leads to increasing charge density When charge density too high: Coulombic explosion Rayleigh limit is reached Analyte molecule Multiply charged droplet www.chm.bris.ac.uk/ms/theory/esi-ionisation.html Charged residue model

Charged amino acids Usually negatively charged at pH 7 Aspartate Glutamate Acidic 3.65 4.24 Usually negatively charged at pH 7 6.00 O Positively/uncharged at pH 7 N N NH Histidine N O + Arginine H3N Lysine Basic 10.8 12.5 Always positively charged

Charges on surface of proteins Red: Negatively charged Blue: Positively charged White: No charge; hydrophobic

Examples ESI of large molecules usually produces multiply charged ions e.g. proteins: Each peak corresponds to the same protein, but with different number of protons attached: Observed ions are [M+nH]n+ 13+ 12+ 11+ 10+ 9+ 8+ 7+ 6+ Charge state series 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z

How to determine the molecular mass of a protein from an ESI-MS spectrum Observed ions have composition [M+nH]n+ Let m1,m2,…, mn : m/z values of the different peaks mn = Its neighbouring peak to the left: mn+1 = Solving both equations for n and M: n = M = n(mn – H) e.g. m2 = 998.1 and m1 = 1060.5 n = 16, and M = 16952 Da 848.7 808.3 771.6 893.3 738.1 942.8 707.4 1 n 1)H] (n [M + 998.1 1060.5 m/z n must be an integer

Deconvolution of ESI mass spectra Deconvoluted spectrum Charge states M = n(mn – H) 3000 30000 Mass (Da)

Quick reminder: average and monoisotopic mass

The distance between isotopic peaks reveals charge state 505.3506 +1 1.00 506.3584 915.4818 915.7363 507.3566 915.9765 +4 915.2247 916.2311 916.4857 0.25 1086.5515 1086.0433 0.51 1086.0444 +2 1087.5529 Jonathan A. Karty 1088.0460

Charge States and distance between isotopic peaks 1695.7 1696.2 1696.7 m/z 0.0 0.5 1.0 1.5 a.i. 0.1 +10

Example beyond molecular mass: Protein folding Calbindin: Calcium binding induces protein folding: increase in charge states with higher m/z (=lower charge, more folded) No Ca2+ excess Ca2+ raw data deconvoluted

Recent developments: ambient mass spectrometry: DESI and DART DESI: Desorption electrospray ionisation DART: Direct analysis in real time Applicable to solids, liquids, and gases No prior sample treatment !

Ionsiation techniques: Summary Ionisation Vola-tile Thermal Size Amount Examples EI Yes Stable Small 1-2 mg organics CI FAB No Medium 0.5-1 mg Polar/ionic organics, organometallics, peptides, biomolecules FD Labile Non-polar organics, organometallics MALDI Large 250 fmol-500 pmol Peptides, proteins, polymers ESI labile 1-300 pmol/ml Polar/ionic organics, peptides, proteins, biomolecules, organometallics, polymers Table adapted from http://www.scs.uiuc.edu/~msweb/SLM530.pdf

Summary: Application ranges of various techniques masspec.scripps.edu/MSHistory/whatisms.php

Self-assessment questions Q1 Describe the two ion formation models in ESI Q2 A positive-ion ESI spectrum shows the following adjacent signals at m/z 4348.8, 4546.5, 4762.9, 5001.0 5264.2. Calculate the molecular mass of the molecule. Q3 The following m/z (979,1040,1109,1189,1280,1387,1512,1664) were obtained by electrospray ionisation of a protein from an aqueous solution. Calculate the molecular mass of the protein within 10 Da. Describe how the mass spectrum would have looked if the same protein had been ionised by MALDI. Q4 What distance between isotopic peaks would you expect for a +12 charge state ? Q5 The following peaks arose from the different isotopes contributing to the ESI mass spectrum resulting from the protonation of a species of relative molecular mass m to reach a charge z.:m/z=848.40, 848.45, 848.50, 848.55, 848.60, 848.65, 848.70. What is the value z and what is the mass of the species giving rise to the peak at m/z 848.55

Tandem MS Peptide/protein identification by MS Lecture 6 Tandem MS Peptide/protein identification by MS

At the end of this session you should be able to explain how structural information can be obtained by Tandem MS and MALDI-TOF/PSD explain how mass spectrometry data can be used to identify known and unknown proteins

Tandem MS (also termed MS2 and MSn) Used for: Identify and quantify compounds in complex mixtures Structure elucidation of unknown compounds Applied in: Proteomics Metabolomics Biomarker discovery De novo protein sequencing

Tandem MS Multistage technique: Ion source MS-1 Activation and fragmentation MS-2 Mass analysis of product ions mass selection of precursor ion MS/MS spectrum Normal spectrum

Tandem MS Fragmentation techniques Collision-induced dissociation (CID): most common Possible with ESI coupled to triple-quad, Ion trap, FT-ICR, and MALDI-TOF Electron Capture Dissociation (ECD): only for multiply charged biopolymers Primarily with FT-ICR Electron-Transfer Dissociation (ETD) Absorption of electromagnetic radiation Not Tandem MS, but useful fragmentation technique: Post-source decay (PSD) combined with MALDI reflectron TOF

Reflectron-TOF and Post-Source decay for MALDI-TOF Field-free region (Some) parent ions fragment in field-free drift region Parent and product ions arrive at reflectron simultaneously (same velocity) Product ions leave Reflectron earlier (smaller Ekin)

Example: MALDI-PSD TOF spectrum of a neuropeptide Parent Ion Necla Birgül, Christoph Weise, Hans-Jürgen Kreienkamp and Dietmar Richter , The EMBO Journal (1999) 18, 5892–5900

Simplified schematic for protein identification from biological samples (“Proteomics”) Cell culture Tissue Biofluid Extraction Complex protein mixture Individual or small sets of proteins Separation Peptide mass mapping Cleavage Peptide-mass fingerprints Peptides MALDI Database search Sequencing (LC-MS/MS) Identified proteins Database search Peptide sequences

Peptide mass mapping/ fingerprinting Makes use of specific cleavage agents Chemical cleavage: e.g. CNBr Digestion with endoproteases (proteolytic enzymes): Trypsin, pepsin, chymotrypsin etc. See exercises

Peptide mass mapping/fingerprinting Protein  Peptides Mass Spectrum Digest Abundance 600 900 1200 1500 1800 m/z Compare Protein Sequence (in database) Theoretical Mass Spectrum Peptide sequences Theoretical Digest QNICPRVNRIVTPCVAYGLGRAPIAPCCRALNDLRFVNTRNLRRAACRCLVGVVNRNPGLRRNPRFQNIPRDCRNTFVRPFWWRPRIQCGRIN NTFVRPFWWRPR IVTPCVAYGLGR CLVGVVNR APIAPCCR FQNIP ... Abundance 600 900 1200 1500 1800 m/z

De novo protein discovery Mass fingerprinting only practicable if protein is already in a database If previously undiscovered protein: Need to sequence Can be done by sequencing peptides

Peptide sequencing: fragmentation rules +NH3―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2― N-terminus C-terminus Three types of bonds along backbone amino alkyl bond alkyl-carbonyl bond amide bond

Peptide sequencing: fragmentation rules b1 c1 R1 R3 R2 R4 NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H x3 y3 z3 Each bond can be broken by fragmentation  Six possible product ions But: peptide bond most likely to break in low energy CID (and MALDI/PSD): Mostly b and y fragments

Peptide fragments generated by low energy CID or PSD NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H N-terminus C-terminus y3 y2 y1 For example: R1 R2 R3 b3: NH2―CH―CO―NH―CH―CO―NH―CH―C=O + R3 R4 y2: +NH3―CH―CO―NH―CH―CO2H m/z values of b ions: residue masses + 1 (+H+) m/z values of y ions: residue masses +17 (OH-) + 2 (2H+) = 19

Example: Peptide analysed by MALDI TOF reflectron and PSD His Leu/ Ile/Asn His Thr Leu/ Ile/Asn Ala Phe Arg Leu/Ile: 113.08 Asn: 114.04  Proposed sequence: HTH[LIN]FA[LIN]R

Self-assessment questions Q1 How are MALDI and ESI used for the identification of proteins ? Q2 Describe how Peptide Fingerprinting works Q3 A MALDI-PSD spectrum of a peptide shows the following y-peaks: 174.8 / 288.3 / 359.0 / 506.1 / 619.6 / 756.0 / 857.6 / 995.2 Find out the masses for amino acids (e.g. at Wikipedia). Calculate the differences between peaks in this spectrum and suggest possible sequences for this peptide

Exercises Exercise 1: Protein cleavage/digestion 1. Go to http://www.expasy.ch/tools/peptidecutter/ 2. In the box, enter ALBU_HUMAN (this is the swissprot name of human serum albumin) - you can also choose a different protein if you like. Sequences and swissprot codes can for example be found in the swissprot database (at www.expasy.ch). 3. Scroll down, and tick the box “only the following selection of enzymes and chemicals”, and then select one chemical or enzyme, which you want to use for cleaving the albumin protein, from the list 4. Scroll back up, and click “Perform” 5. Inspect the output. How many times is albumin cleaved by your chosen cleavage agent ? Find out what the specificity of your cleavage agent is.

Exercise 2: Calculation of molecular masses of proteins and peptides 1. Copy a peptide fragment from the output of Exercise 1 (or make one up yourself), go to http://www.expasy.ch/tools/protparam.html and paste your sequence into the appropriate box (the large one). 2. Click “Compute Parameters”. 3. Inspect the output. What is the molecular formula of the peptide ? How many positively and negatively charged side-chains does your peptide have ? What charge would it have at pH 7 ?

Exercise 3. Average and monoisotopic masses 1. Copy the molecular formula (or the one-letter code sequence) of the peptide from exercise 2, and go to http://education.expasy.org/student_projects/isotopident/htdocs/ 2. Paste the formula or sequence in the appropriate box. Make sure that you have selected the correct “Type of composition” (e.g. “chemical formula”) in the respective pulldown menu. 3. Click “Submit query”. 4. Inspect the output. How many isotopic peaks are there? Which is the most abundant peak ? What are the monoisotopic and the average masses ?