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Lecture Four: "Bigger, Better, Faster, Stronger: the Cutting Edge" Advanced MS instruments Advanced MS techniques.

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Presentation on theme: "Lecture Four: "Bigger, Better, Faster, Stronger: the Cutting Edge" Advanced MS instruments Advanced MS techniques."— Presentation transcript:

1 Lecture Four: "Bigger, Better, Faster, Stronger: the Cutting Edge" Advanced MS instruments Advanced MS techniques

2 Electrospray ionization (ESI)

3 Comparing ionization techniques MALDI –Bias for polar/charged peptides –Relatively salt tolerant –Suitable for complex mixes –Must be “offline” –Relatively low res due to desorption velocity –Matrix <1000 m/z –Photo crosslinking and degradation –Nobel Prize in Chemistry, 2002 ESI –Bias for nonpolar peptides. –Very salt sensitive –Low tolerance for very complex mixes –Requires expertise and $’s to run and maintain –Analyses can be coupled to µLC –> sequence coverage –Nobel Prize in Chemistry, 2002

4 quadrupole mass analyzer / filter

5 Mass filtering and “scanning” Differs from TOF The quad is a “mass filter” At a specified voltage frequencies, only ions w/ certain m/z’s will reach the detector. The voltage relationship is varied during the real- time analysis, thus “scanning” a constant beam of ions.

6 Tandem mass spectrometers Two mass spectrometers linked end to end Ions are passed through the first MS, then again through the second MS Increased resolution Decay between MS sectors

7 Triple-sector Quadrupole (Q/Q)

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9 MS/MS In PMF, the protein is cut by a protease, then the fragments are sized. In MS/MS, the proteolytic fragments are broken further by collision during the actual MS process.

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11 Triple-sector Quadrupole (Q/Q)

12 Mass differences = f (sequence)

13 Using MS/MS data Observed spectra masses are used to search database of theoretical fragment masses –Requires that peptide sequence in d-base –Can be used to complement PMF searching, or independent –Peptide’s intact mass is used to filter d-base like intact protein MW of PMF search. Attempt to solve peptide sequence based on mass differences between fragments –“de novo” sequencing of novel protein sequences (no d-base) – not trivial, but software continues to advance

14 Types of Scans I Product / daughter ion scans –Like PSD –Sector #1: filter for one specific precursor / parent –Sector #2: scan the fragments / products / daughters

15 Types of Scans II Dynamic exclusion –Automation of product ion scanning process –Initial analyses of ion beam ID's the predominant parent ions, and collects MS 2 of the daughters. –Subsequent analyses exclude this parent, and instead "drill down" to analyze the less abundant ions.

16 Types of Scans III Precursor / parent scans –Sector #2: set to filter / detect one product/daughter mass –Sector #1: scan to ID the parent responsible

17 Types of Scans IV Neutral loss –Two sectors scan in tandem –These tandem scans are offset by a specific mass (e.g., 80 amu) –Attempt to ID specific parent / daughter pairs (e.g.,  = 80 amu: loss of phosphate from pS/pT)

18 Ion Trap mass spectrometers I Ions are trapped in stable trajectories, and thus accumulated in a magnetic / electrostatic “bottle.” Varying the fields allows specific m/z's to ejected to the detector. Great sensitivity, mediocre resolution.

19 Ion Trap mass spectrometers II Alternatively, the fields can be varied to destabilize the trajectory of specific m/z's, causing them to be quenched on the electrodes. Subsequently, the remaining specific m/z ions can be ejected

20 Ion Trap mass spectrometers III To get really fancy, the fields can be varied to quench the ions that are not of interest. Then, accelerate the remaining interesting (specific m/z ions), causing high-energy collisions and fragmentation. Now, eject those fragments as a sizing scan.

21 Ion Trap mass spectrometers IV Now…do it again (MS 3 ). This time, quench everything except that interesting daughter. Now, fragment her, and then eject her fragments as a sizing scan. And again and again… These sequential fragmentations are like physical "sectors"

22 Q's v.s. traps Quadrupole –"beam" technique filter the beam let "m/z = x" past "filter input" –10 -6 sec per acquistion –>>y ions, << b ions –Low mass frags present in MS 2 –MS 2 limit Traps –Trapping interrupts beam Ions in beam are trapped Subsequent release of "m/z = x" –10 -4 sec per acquisition –b & y ions –"Rule of Thirds" Ions <1/3 x mass parent not seen offset by presence of b ions –Trapping allows sequential release of frags MS n "n" up to 10

23 Linear ion traps Newest innovation in trapping Enhanced trap capacity –Greater sensitivity Enhanced trap fill rate –Better coverage, because faster cycle time allows analysis of more peptides in a given nLC peak.

24 Ion cyclotron resonance A mixture of ions are trapped in a “EM field bottle,” where an externally applied magnetic field causes them to move in circular orbits. The radii of the orbits correspond to the ions’ m/z.

25 Ion cyclotron resonance Then the circling ions are pulsed with an oscillating electrical field (a radio frequency). If the frequency of the field matches that of the circling ions, they adsorb that energy, and their circular radius & speed will increase. Only specific m/z’s are thus accelerated.

26 Ion cyclotron resonance The ion’s new orbit and speed are maintained after the pulse.

27 Ion cyclotron resonance As the ions pass each member of a pair of charged electrodes, an electrical current is produced.

28 FT-ICR MS The electrical current thus produced is interpreted mathematically via the “Fourier Transformation”

29 Fourier-transformation Isotope Cyclotron Resonance MS

30 FT-MS Low fmole sensitivity R = 800,000! ‘twas strictly experimental four years ago. Now commercially available.

31 Surface Enhanced Laser/ Desorption Ionization (SELDI) “protein chip” technology by Ciphergen Each spot is a different “affinity patch” to “semi-purify” proteins Followed by whole- protein MALDI-TOF

32 SELDI: Finding changes in protein patterns that correlate to certain disease states diagnostics prognostics replacement of other protein separation systems for protein discovery interpret w/ advanced software example

33 SELDI (cont’d) Start w/ coated plates. Bind complex cell lysate. Wash. Overlay matrix. Shoot MALDI-TOF.

34 SELDI Example: in vivo inhalation of house dust allergen cause 3 new proteins to appear in the broncho- alveolar lavage fluid of mice. % Garcia and Brower, John Hopkins

35 Raw Patterning Like SELDI, but without Ciphergen chips. MALDI AND ESI What small proteins (masses) are present in unfractionated lysate? “Genotyping, “ pathogen-specific small protein “fingerprints,” serum profiling, etc. CDC, DOD, NIH, etc.

36 Direct Analysis in Real Time (DART) Beam a stream of excited helium plasma at the sample--> to the MS. Atmospheric pressure Fast Forensics and security applications

37 Secondary Ion Mass Spectrometry (Imaging Mass Spectrometry) An ionizing beam is directed at the sample. The resultant secondary ions are captured and analyzed. Mass of secondary ion ~ color (mass filter) Very important in materials fabrication (e.g., semiconductors). Images % N. Winograd et al.

38 Combining liquid chromatography and mass spec Chromatographer: “MS is a fancy detector.” MS jock: “Chromatography is a fancy sample prep method. LC separations and MS analyses can be directly coupled for real time analysis. Tiny LC scale (nano- to microliters; nLC, capillary LC).

39 LC-MS Samples are digested with trypsin

40 LC-MS …and the resultant peptides are separated by µHPLC…

41 LC-MS …on the basis of their different affinities for the stationary column packing …

42 LC-MS …and eluted from the column …

43 LC-MS … into the electrospray ionization source of the MS …

44 …for MS/MS!

45 What have we accomplished w/ LC-MS/MS? Limitations of ESI-MS are addressed: –Samples contaminated by salt –Complex samples Peptide separations and MS are “on line,” with no intervening fraction collection and sample handling. We are now staged for high-throughput multi-dimensional LC-MS/MS!

46 Microfluidic Chips / ESI Sources Laser etching and polymer lamination create micro- channels (like semi- conductor manufacturing).Laser etching and polymer lamination create micro- channels (like semi- conductor manufacturing). Channels can be coated (“packed”) with chromo media.Channels can be coated (“packed”) with chromo media. Multiple “micro-columns” per chip.Multiple “micro-columns” per chip. Various strategies for switching valves and mobilizing fluid phase.Various strategies for switching valves and mobilizing fluid phase. Fluid path terminates with ESI nozzle, bolted to ESI- MS instrument.Fluid path terminates with ESI nozzle, bolted to ESI- MS instrument.

47 Yin et al. (2005) Anal Chem. 77:537

48 Results for Tryptic digest of BSA Five chromatograms, ( nL / min).Five chromatograms, ( nL / min). X axis is time / flow/ volume of solvent.X axis is time / flow/ volume of solvent. Y axis is intensity of base peak in mass spectra (individual peptides).Y axis is intensity of base peak in mass spectra (individual peptides). Yin et al. (2005) Anal Chem. 77:537

49 high-throughput Multi-Dimensional Protein Identification Technique (MuDPIT) Digest the whole proteome Separate the peptides by two-dimensional chromatography –C18 reverse-phase µHPLC, then –ion exhange (or…) ESI/MS/MS Bioinformatics & databases

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51 MuDPIT v.s. 2-DE Mudpit nicely addresses 2-DE limitations –< amount of protein –naughty proteins –can be highly automated, limited “hands on” But.. –not quantitative –very computer intensive –quite challenging

52 The Heavenly Pair: LS-MS/MS & 1-D gels 1-DE gels of protein complexes often fail to resolve all components. However, MS/MS will tolerate a mix. You get to skip the 2-DE!!! > sensitivity and

53 The challenge of gel-less proteomics: quantitation Why is quantitation important? Isotope-Coded Affinity Tagging iTRAQ and AQUA

54 The challenge of gel-less proteomics: quantitation Why is quantitation important? Isotope-Coded Affinity Tagging iTRAQ and AQUA

55 Quantitation via ICAT et al. The whole proteome is digested. Peptides containing Cys are chemically labeled with Biotin

56 ICAT (cont’d) The mix of peptides is passed through a streptavidin column Biotinylated peptides stick… …and are subsequently eluted. The peptide population is simplified.

57 The elegant twist: remember isotopes? Isotopes have an extra neutron. Isotopic molecules have more mass than their non-isotopic counterparts. The MS can see this difference. Different versions of the tag are used for two different tissues, one tag w/o isotopes, and one tag with.

58 ICAT Peptides from two tissues –cleaved and labeled in separate tubes. –one tissue gets “no isotope” tag –other gets tag “w/ isotope” Mix, separate by chromo, and analyze by MS. –specific mass difference –peak heights are now quantitatively representative (why?)

59 The challenge of gel-less proteomics: quantitation Why is quantitation important? Isotope-Coded Affinity Tagging iTRAQ and AQUA

60 Sigma’s AQUA peptides for Absolute Quantitation Study your protein’s sequence and PMF spectra. Select a good “tryptic peptide” sequence. Chemically synthesize and quantitate a heavy-isotope version.

61 Sigma’s AQUA peptides for Absolute Quantitation Add a known quantity of this heavy-labeled tracer to your sample.

62 Sigma’s AQUA peptides for Absolute Quantitation Digest with trypsin. Run MuDPIT

63 AQUA MS Analysis, focused on your peptide. Heavy-labeled peptide serves as relative and absolute internal standard.

64 ABI’s iTRAQ system Isobaric tags: four reactive tagging agents. Tags have same net mass, distributed between “reporter” and “balance” segments. Tags also drive ionization and CAF.

65 … but MS/MS (B) unmasks fragments of tag that are quantitatively representative (C) while still allowing clean peptide MS/MS (D&E). Modified pooled peptides look like a single peptide in MS mode…

66 Hydrogen/Deuterium Exchange A method for probing protein structure. Overcomes limitations of NMR and X-ray –sensitive –dynamic –doesn’t require crystals Done w/ MS

67 Consider the amide hydrogen...

68 Amide hydrogens Relatively slow exchange with hydrogens of water, due to the partial double-bond nature of the amide bond.

69 …amide hydrogens... …unless they are involved in hydrogen bonds. In proteins, they often are!

70 H/D exchange... But proteins are dynamic molecules that “breath.” Changes in protein conformation, ligand binding, or protein- protein interactions alter the hydrogen bond network.

71 H/D exchange... But proteins are dynamic molecules that “breath.” Changes in protein conformation, ligand binding, or protein- protein interactions alter the hydrogen bond network.

72 H/D exchange... But proteins are dynamic molecules that “breath.” Changes in protein conformation, ligand binding, or protein- protein interactions alter the hydrogen bond network.

73 H/D X  Experimental Flow Label intact protein with D 2 O in the presence versus absence of ligand. Quench exchange with low temp & pH Cut with pepsin under quench conditions Rapid MS analysis using chilled plates and speed-vac drying Analyze peptides for centroid of peak series (average mass for the peptide). Look for retardation of labeling

74 H/D exchange... Deuterium oxide is “heavy water,” wherein the hydrogen is replaced by deuterons (heavy protons). Hmmm…

75 H/D exchange... Replacement of the amide H with D will result in a mass shift. The rate and magnitude of this shift reflects the H-bonding environment. If folding or ligand binding alters this environment...

76 End of Lecture Four Questions? The instructor and hardware belong to you for the next two days.


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