Outline

1
Outline

• Introduction
• What's it all about
• Course Mechanics
• From Genomics to Proteomics
• Protein Separation
• Protein Identification
• Mass Spectrometry
• Fundamentals
• Protein Chemistry
• Ionization Techniques
• Fragmentation techniques
• Mass Analyzers
• CID MS/MS Interpretation
• Peptide Fragmentation Chemistry
• Interpretation of Spectra

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Analyzing Spectra
3
Identifying Proteins with MS

• There are three main approaches for identifying
  proteins.
• Peptide sequence tag
• Database search
• Using de novo algorithm to build peptide sequence.

4
Identifying Proteins with MSUsing Peptide
Sequence Tags

• In tandem mass spectrometry, peptides are
  fragmented.
• The location and identity of some amino acid in
  these fragments can be determined by the spacing
  of these fragments of the mass of that amino
  acid.
• Since there are 20 kinds of amino acids
• Some combinations of amino acids are sufficient
  to identify protein in database
• Or at least help greatly to reduce the number of
  matched peptide sequence
• Called peptide sequence tags
• Mann and Wilm used a combination of a partial de
  novo algorithm and a database search to implement
  this method
• The limitation of this method is that if the
  peptide sequence tag is not suitably selected
• It will produce both false positives
• And false negatives.

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Identifying Proteins with MSUsing Database
Search

• During the process of mass spectrometry, the
  related mass spectra represent the experimental
  spectrum.
• For all sequences in a protein database or
  genomic database
• We construct a theoretical mass spectrum.
• An exhaustive search is then used to compare
  experimental spectrum and all these theoretical
  ones.
• This method has following limitations
• Post-translational modifications will reduce the
  accuracy of identification
• With accumulation of protein sequences, this
  method can be slow.
• This method will not find new proteins that are
  not in the database.

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Identifying Proteins with MSBy de novo Sequencing

• First generate a spectrum graph.
• Then attempt to find what is called a feasible
  path through this graph.
• The feasible path corresponds to a valid peptide
  sequence.

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De novo Peptide Sequencing
Sequence
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Generating a Theoretical Spectrum for a Database
Search
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Generating a Theoretical Spectrum for a Database
Search
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Generating a Theoretical Spectrum for a Database
Search
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Building a Spectrum Graph for de novo Sequencing

• How to calculate masses of b and y ions
• How to create vertices (from masses)
• How to create edges (from mass differences)
• How to score paths
• How to find best path

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Protein Backbone Plus Water at Termini
H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-OH
Ri-1
Ri
Ri1
C-terminus
N-terminus
AA residuei-1
AA residuei1
AA residuei
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y Ion Mass

• C terminal peptide has an extra OH
• N terminal peptide has an extra H
• Must add
• 18 Da. for the H20
• 1 Da. for the proton
• So, add the residue masses and add 19
• Assuming singly charged

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b Ion Mass

• b ion is different
• C terminal peptide forms cyclic structure (see
  next slide)
• Thus, has no extra OH
• In fact, the N on the final residue (on the C
  terminal end) has an extra covalent bond with the
  backbone Carbons
• Thus, it doesnt have an H
• Thus, the residue is 1 Da less
• (see next slide again)
• N terminal peptide has an extra Hydrogen
• Must add
• 1 Da. for the N terminal H
• 1 Da. for the proton
• -1 Da for the loss of the H on the final residue
• So, add the residue masses and add 1
• Assuming singly charged

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b and y Ion Chemistry
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S E Q U E N C E
b
Mass/Charge (M/Z)
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a
S E Q U E N C E
Mass/Charge (M/Z)
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a is an ion type shift in b
S E Q U E N C E
Mass/Charge (M/Z)
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y
E C N E U Q E S
Mass/Charge (M/Z)
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y with corresponding intensities
E C N E U Q E S
Intensity
Mass/Charge (M/Z)
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Intensity
Mass/Charge (M/Z)
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Intensity
Mass/Charge (M/Z)
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noise
Mass/Charge (M/Z)
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MS/MS Spectrum
Intensity
Mass/Charge (M/z)
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Mass Differences Correspond to Amino Acids
u
q
e
e
q
s
u
e
n
n
c
e
e
e
q
c
s
n
e
s
u
e
c
e
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de novo Sequencing from the C terminus (1/4)

• To begin sequencing a tryptic peptide, Assume
  that the C-terminus of the peptide is either
  lysine or arginine.
• This assumption is usually true except for
• Tryptic peptides derived from non-tryptic
  cleavage
• Due to contaminating chymotryptic activity
• Or tryptic peptides encompassing the C-terminus
  of the original protein
• Where the C-terminal residue of the protein is
  not lysine or arginine.
• The y1 ion is calculated by adding 19.018 Da t o
  the residue masses of lysine and arginine
• Three hydrogens and one oxygen
• H20 plus proton
• Lysine
• 128.095 19.018 147.113 Da
• Arginine
• 156.1011 19.018 175.119 Da
• If either mass is present, make a note.

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de novo Sequencing from the C terminus (2/4)

• On ion traps, the y1 ions will be below the mass
  cutoff
• The corresponding high m/z b-type ion is often
  found, though
• This b ion contains all of the residues except
  the arginine or lysine at the C-terminus.
• These b ions are calculated by
• Subtract 17.002 Da from the precursor mass
• 17.002 Da One oxygen and one hydrogen
  Water loss plus proton gain
• Subtract the residue mass of arginine or lysine.
• If this b ion is found, make a note of it.

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de novo Sequencing from the C terminus (3/4)

• If a y1 ion for lysine or arginine is found
• Get peak corresponding to higher product ion
  masses
• Subtract the y1 mass
• Check in the residue mass table to see if any
  mass differences correspond to an amino acid.
• If any differences equate to an amino acid
  residue mass
• Make a note of what each putative y2 ion might be
• Also subtract each possible residue mass from the
  high mass b ions
• Recall these correspond to loss of arginine or
  lysine from precusor
• high m/z b ions rarely seen for ion traps or
  QTOFs
• Proceed to the y3 ion and higher
• For each, check to see if the corresponding high
  m/z b-type ion is present.
• Eventually as amino acid residue masses are added
  to the y ion series, it passes the b ion series.

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de novo Sequencing from the C terminus (4/4)

• Favored partial sequence appear to have both
• High m/z b ions
• Corresponding y ions.
• Eventually, you might get a complete sequence for
  the peptide
• Hypothesized sequence should have a calculated
  mass that equals the observed precursor mass
• Within the error tolerance of the peptide mass
  measurement
• Often you cannot get a complete sequence all the
  way to the N-terminus
• It is common for a CID spectrum to lack
  fragmentations between the first and second amino
  acids at the N-terminus.
• Therefore, no b1 ion observed
• The N-terminus of this proposed has the combined
  residue mass of the first two amino acids.
• Make sure that this unsequenced mass at the
  N-terminus corresponds to the sum of two amino
  acid residue masses.
• For example, an unsequenced N-terminal mass of
  150 Da is not possible in the absence of the
  additional mass of a post-translational
  modification.

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de novo Sequencing from the Middle (1/4)

• Procedure
• Get some partial sequence from the middle of the
  peptide
• Try to connect this partial sequence to the
  peptide N-terminus.
• Proceed to C terminus.
• For Qtof or Triple Quads there is a short stretch
  of fairly intense ions at a m/z greater than the
  precursor m/z
• The mass differences between these ions in the
  series correspond to amino acid residue masses
• These are the so-called sequence tags introduced
  by Matthias Mann
• In principal, one does not know if these are
  b-type or y-type ions
• And hence, whether the partial sequence goes
  forward or backwards
• For Qtof and triple quad tryptic peptides it is
  usually safe to guess that this is a partial y
  ion series.
• The precursor must be doubly or triply charged

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de novo Sequencing from the Middle (2/4)

• Take the peptide mass 2.016 Da, and subtract the
  highest mass ion in this series
• This is the mass of two protons
• The peptide mass has no ions, but the b and y
  ions each have one.
• This mass difference would correspond to a
  hypothetical lower mass b ion.
• (Precursor Mass 2) - (y ion with proton) (b
  ion with proton)
• Often times a y ion series will encompass all but
  the two N-terminal amino acids
• Then the mass difference between that y ion and
  the precursor mass 2 corresponds to a b2 ion.
• If that b2 ion is present, check to see if there
  is another ion 27.995 Da lower
• Which would possibly be the matching a2 ion.

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de novo Sequencing from the Middle (3/4)

• If all of these ions are found
• The putative high m/z y ion
• Plus the alleged low m/z b
• Plus the a ion
• Then its most likely a real peptide ion.
• If you are lucky, the high m/z y-type ion series
  extends all the way to the N-terminus
• In which case this mass difference corresponds to
  b1 ion
• An amino acid residue mass plus a hydrogen
• Don't bother looking for a b1 ion they don't
  exist.
• If a partial y ion series is found, then try to
  identify the low mass b ion series that
  corresponds to the high m/z y ion series.

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de novo Sequencing from the Middle (4/4)

• For Qtof and triple quad tryptic peptides, the b
  ions usually decrease in intensity to negligible
  values at the high end
• At the same time the corresponding y ions are
  going into the low m/z end of the spectrum, where
  its harder to see.
• This portion of the spectrum usually contains
  many more fragment ions of different type
• Immonium ions
• b ions
• a ions
• y ions
• Other charged fragments
• Keep trying to connect the high m/z y-type ion
  series until you reach a y1 ion for the
  C-terminal lysine or arginine
• 147.113 Da or 175.119 Da, respectively

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Peak Tips What to Look for in a Spectrum

• Look for the tallest peak in the spectrum above
  200 mass units.
• The low end of a spectrum can often be confounded
  with solvent noise.
• Then look for peaks that are roughly double or
  half the mass.
• This may tell you whether there are multiply
  charged species present which can help with mass
  determination.   
• Counts. 
• Resist interpreting meager spectra.
• If you know that on a particular day that 1.0106
  is a respectable, reliable signal then you will
  know that a spectrum that tops out at 1.0104
  counts (or at background) may not be a reliable
  spectrum to interpret.    

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Peak Tips What to Look for in a Spectrum

• The quality of the spectrum is important.
• You will waste valuable time interpreting a low
  quality spectrum. 
• If you are not happy with the quality of the
  spectrum try averaging several or many low level
  spectra to obtain a better quality "averaged mass
  spectrum." 
• Compare this to a background spectrum to see if
  the peaks really stand out.  
• Reproducibility is important. 
• The peak must be consistent to be considered a
  relevant peak.
• You should not lend credence to "one scan
  wonders."  

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Peak Tips What to Look for in a Spectrum

• Determining the charge state of a peak when only
  one peak is obvious
• A molecule will often have adduct ions associated
  with it other than hydrogen . 
• Look for sodium or ammonium adducts. 
• These adducts can often give you a hint as to the
  charge state of a peak. 
• For example if there is only one major species in
  a spectrum, look for the sodium adduct following
  that peak. 
• If it is a singly charged species the sodium
  adduct will be found at 22 mass units higher
  than the MH peak. 
• If the peak is doubly charged the adduct will
  appear at 11 mass units.

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Isotopes

• If the mass spectrometer you are working with has
  sufficient resolution, look at the isotopes
• A singly charged ion will show isotopic peaks
  that differ by 1 mass unit
• A doubly charged ion will show peaks that differ
  by 0.5 mass units and so on.
• This is another way to deduce the charge state of
  a peak and thus the mas. 

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Frequently Asked Questions

• Q How can I tell if a peak is real?
• A Wow, this is some question. 
• All peaks are real. 
• In an LC/MS run, we look for peaks that reoccur
  in multiple adjacent scans (spectra) but not in
  every scan. 
• If the peak occurs in every scan it may be a
  background peak.
• It is possible to get system noise or spikes that
  only occur in one scan or sporadically these are
  most likely electronic or some other form of
  system noise. 

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Frequently Asked Questions

• Q How can I be sure of the identity of a peak.
• A Well, a mass is just a mass and many compounds
  have isobaric mass so you can't be sure from just
  a mass. 
• In the old days we would
• Perform an enzymatic digest on a protein
• Run an LC/MS peptide map and
• Match up the mass with the theoretical fragments.
  
• Today the bar is rightly higher and we go one
  step further in the identification
• We take the peak through a fragmentation
• And match up the fragment masses with the
  theoretical CID fragment masses for that peptide.
  
• This gives us a positive ID.
• Another overlooked component in LC/MS
• The correlation of mass and LC retention time. 
• This is part of what makes LC/MS so powerful
• If the retention time of a molecule has been
  previously characterized this information can be
  linked with the mass information for a positive
  ID.
• If you are characterizing a new molecule
• Try modifying the molecule to see if you can
  modify the mass. 
• Try an enzyme digest if the unknown is a protein

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Frequently Asked Questions

• Q How can I differentiate a compound at one mass
  from another at twice the mass? 
• For example a compound with mass 1000 will
  display peaks at m/z 1001 and 501,
• (1000 1) / 1
• (1000 2) / 2
• A compound with mass 2000 may display peaks at
  m/z 2001, 1001, 667.7 and 501. 
• (2000 1) / 1
• (2000 2) / 2
• (2000 3) / 3
• (2000 4) / 4
• The mass determination can further be confounded
  if the peptide at 1000 forms dimers during the
  electrospray process.

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Frequently Asked Questions

• A
• The peak envelope does not skip peaks
• For example the 2000 mass even if it does not
  have an obvious peak at 2001 it should have the
  667.7 peak between the 1001 and 501 peaks.
• Also try to determine the charge state of the
  ions from the adducts or from the isotopes. 
• This will tell you what the mass of the compound
  is.
• Dimer formation can be a major problem in some
  analyses.
• Try to reduce the concentration of the analyte.
• Often if the concentration is too high dimers
  will be observed in the spectrum. 
• Also dimers can be reduced by changing some of
  the setting on the mass spectrometer.

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Frequently Asked Questions

• With the peak envelope of larger molecules
  (10kDa) look for smooth peak distributions. 
• The peak distribution should have a smooth bell
  shaped curve appearance, sometimes trailing off
  to the right.
• The peak to peak relationship should be
  predictable
• If one observes an alternating pattern of peak
  intensities this may be a clue to a co-eluting
  dimer. 

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Example
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Example
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Example
389.9 (2/2) 2 779.8 - 2 777.8
(777.8 2) - 518.3 261.5 261.2
27.995 233.2
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Example
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Example
389.9 (2/2) 2 779.8 - 2 777.8
(777.8 2) 431.2 348.6
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Example
348.0 - 27.995 320.0
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Example
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Example
389.9 (2/2) 2 779.8 - 2 777.8
(777.8 2) 303.1 476.7
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Example
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Example
389.9 (2/2) 2 779.8 - 2 777.8
(777.8 2) 204.1 575.7
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Example
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Example
389.9 (2/2) 2 779.8 - 2 777.8
(777.8 2) 147.1 632.7
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• Kinter Sherman pg. 93

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