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Published on October 15, 2007

Author: Malden

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CSE182-L7:  CSE182-L7 Protein sequencing and Mass Spectrometry Announcements:  Announcements Midterm 1: Nov 1, in class. Assignment 2: Online, due October 20. Trivia Quiz:  Trivia Quiz What research won the Nobel prize in Chemistry in 2004? In 2002? How are Proteins Sequenced? Mass Spec 101::  How are Proteins Sequenced? Mass Spec 101: Nobel Citation 2002:  Nobel Citation 2002 Nobel Citation, 2002:  Nobel Citation, 2002 Mass Spectrometry:  Mass Spectrometry Sample Preparation :  Sample Preparation Single Stage MS:  Single Stage MS Mass Spectrometry LC-MS: 1 MS spectrum / second Tandem MS:  Tandem MS Secondary Fragmentation Ionized parent peptide The peptide backbone:  The peptide backbone H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH Ri-1 Ri Ri+1 AA residuei-1 AA residuei AA residuei+1 N-terminus C-terminus The peptide backbone breaks to form fragments with characteristic masses. Ionization:  Ionization H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH Ri-1 Ri Ri+1 AA residuei-1 AA residuei AA residuei+1 N-terminus C-terminus The peptide backbone breaks to form fragments with characteristic masses. Ionized parent peptide H+ Fragment ion generation:  Fragment ion generation H...-HN-CH-CO NH-CH-CO-NH-CH-CO-…OH Ri-1 Ri Ri+1 AA residuei-1 AA residuei AA residuei+1 N-terminus C-terminus The peptide backbone breaks to form fragments with characteristic masses. Ionized peptide fragment H+ Tandem MS for Peptide ID:  Tandem MS for Peptide ID 147 K 1166 L 260 1020 E 389 907 D 504 778 E 633 663 E 762 534 L 875 405 F 1022 292 G 1080 145 S 1166 88 y ions b ions 100 0 250 500 750 1000 [M+2H]2+ m/z % Intensity Peak Assignment:  Peak Assignment 147 K 1166 L 260 1020 E 389 907 D 504 778 E 633 663 E 762 534 L 875 405 F 1022 292 G 1080 145 S 1166 88 y ions b ions 100 0 250 500 750 1000 y2 y3 y4 y5 y6 y7 b3 b4 b5 b8 b9 [M+2H]2+ b6 b7 y9 y8 m/z % Intensity Peak assignment implies Sequence (Residue tag) Reconstruction! Database Searching for peptide ID:  Database Searching for peptide ID For every peptide from a database Generate a hypothetical spectrum Compute a correlation between observed and experimental spectra Choose the best Database searching is very powerful and is the de facto standard for MS. Sequest, Mascot, and many others Spectra: the real story:  Spectra: the real story Noise Peaks Ions, not prefixes & suffixes Mass to charge ratio, and not mass Multiply charged ions Isotope patterns, not single peaks Peptide fragmentation possibilities (ion types) :  Peptide fragmentation possibilities (ion types) Ion types, and offsets:  Ion types, and offsets P = prefix residue mass S = Suffix residue mass b-ions = P+1 y-ions = S+19 a-ions = P-27 Mass-Charge ratio:  Mass-Charge ratio The X-axis is (M+Z)/Z Z=1 implies that peak is at M+1 Z=2 implies that peak is at (M+2)/2 M=1000, Z=2, peak position is at 501 Suppose you see a peak at 501. Is the mass 500, or is it 1000? Isotopic peaks:  Isotopic peaks Ex: Consider peptide SAM Mass = 308.12802 You should see: Instead, you see 308.13 308.13 310.13 Isotopes:  Isotopes C-12 is the most common. Suppose C-13 occurs with probability 1% EX: SAM Composition: C11 H22 N3 O5 S1 What is the probability that you will see a single C-13? Note that C,S,O,N all have isotopes. Can you compute the isotopic distribution? All atoms have isotopes:  All atoms have isotopes Isotopes of atoms O16,18, C-12,13, S32,34…. Each isotope has a frequency of occurrence If a molecule (peptide) has a single copy of C-13, that will shift its peak by 1 Da With multiple copies of a peptide, we have a distribution of intensities over a range of masses (Isotopic profile). How can you compute the isotopic profile of a peak? Isotope Calculation:  Isotope Calculation Denote: Nc : number of carbon atoms in the peptide Pc : probability of occurrence of C-13 (~1%) Then +1 Nc=200 Isotope Calculation Example :  Isotope Calculation Example Suppose we consider Nitrogen, and Carbon NN: number of Nitrogen atoms PN: probability of occurrence of N-15 Pr(peak at M) Pr(peak at M+1)? Pr(peak at M+2)? How do we generalize? How can we handle Oxygen (O-16,18)? General isotope computation:  General isotope computation Definition: Let pi,a be the abundance of the isotope with mass i Da above the least mass Ex: P0,C : abundance of C-12, P2,O: O-18 etc. Characteristic polynomial Prob{M+i}: coefficient of xi in (x) (a binomial convolution) Isotopic Profile Application:  Isotopic Profile Application In DxMS, hydrogen atoms are exchanged with deuterium The rate of exchange indicates how buried the peptide is (in folded state) Consider the observed characteristic polynomial of the isotope profile t1, t2, at various time points. Then The estimates of p1,H can be obtained by a deconvolution Such estimates at various time points should give the rate of incorporation of Deuterium, and therefore, the accessibility. Quiz:  Quiz How can you determine the charge on a peptide? Difference between the first and second isotope peak is 1/Z Proposal: Given a mass, predict a composition, and the isotopic profile Do a ‘goodness of fit’ test to isolate the peaks corresponding to the isotope Compute the difference Tandem MS summary:  Tandem MS summary The basics of peptide ID using tandem MS is simple. Correlate experimental with theoretical spectra In practice, there might be many confounding problems. A toolkit that resolves some of these problems will be useful. MS Quiz::  MS Quiz: Why aren’t all tandem MS peaks of the same intensity? Do the intensities for a peptide vary from spectrum to spectrum? De novo interpretation of mass spectra:  De novo interpretation of mass spectra The so called de novo algorithms focus exclusively on the D module. There is no database (I/F). Limited scoring and validation Computing possible prefixes:  Computing possible prefixes We know the parent mass M=401. Consider a mass value 88 Assume that it is a b-ion, or a y-ion If b-ion, it corresponds to a prefix of the peptide with residue mass 88-1 = 87. If y-ion, y=M-P+19. Therefore the prefix has mass P=M-y+19= 401-88+19=332 Compute all possible Prefix Residue Masses (PRM) for all ions. Putative Prefix Masses:  Putative Prefix Masses Prefix Mass M=401 b y 88 87 332 145 144 275 147 146 273 276 275 144 S G E K 0 87 144 273 401 Only a subset of the prefix masses are correct. The correct mass values form a ladder of amino-acid residues Spectral Graph:  Spectral Graph Each prefix residue mass (PRM) corresponds to a node. Two nodes are connected by an edge if the mass difference is a residue mass. A path in the graph is a de novo interpretation of the spectrum 87 144 G Spectral Graph:  Spectral Graph Each peak, when assigned to a prefix/suffix ion type generates a unique prefix residue mass. Spectral graph: Each node u defines a putative prefix residue M(u). (u,v) in E if M(v)-M(u) is the residue mass of an a.a. (tag) or 0. Paths in the spectral graph correspond to a interpretation Re-defining de novo interpretation:  Re-defining de novo interpretation Find a subset of nodes in spectral graph s.t. 0, M are included Each peak contributes at most one node (interpretation)(*) Each adjacent pair (when sorted by mass) is connected by an edge (valid residue mass) An appropriate objective function (ex: the number of peaks interpreted) is maximized 87 144 G Two problems:  Two problems Too many nodes. Only a small fraction are correspond to b/y ions (leading to true PRMs) (learning problem) Even if the b/y ions were correctly predicted, each peak generates multiple possibilities, only one of which is correct. We need to find a path that uses each peak only once (algorithmic problem). In general, the forbidden pairs problem is NP-hard However,..:  However,.. The b,y ions have a special non-interleaving property Consider pairs (b1,y1), (b2,y2) If (b1 < b2), then y1 > y2 Non-Intersecting Forbidden pairs:  Non-Intersecting Forbidden pairs 300 S G E K If we consider only b,y ions, ‘forbidden’ node pairs are non-intersecting, The de novo problem can be solved efficiently using a dynamic programming technique. 87 332 The forbidden pairs method:  The forbidden pairs method There may be many paths that avoid forbidden pairs. We choose a path that maximizes an objective function, EX: the number of peaks interpreted The forbidden pairs method:  The forbidden pairs method Sort the PRMs according to increasing mass values. For each node u, f(u) represents the forbidden pair Let m(u) denote the mass value of the PRM. u f(u) D.P. for forbidden pairs:  D.P. for forbidden pairs Consider all pairs u,v m[u] <= M/2, m[v] >M/2 Define S(u,v) as the best score of a forbidden pair path from 0->u, v->M Is it sufficient to compute S(u,v) for all u,v? 300 100 400 200 0 87 332 u v D.P. for forbidden pairs:  D.P. for forbidden pairs Note that the best interpretation is given by 300 100 400 200 0 87 332 u v D.P. for forbidden pairs:  D.P. for forbidden pairs Note that we have one of two cases. Either u < f(v) (and f(u) > v) Or, u > f(v) (and f(u) < v) Case 1. Extend u, do not touch f(v) 300 100 400 200 0 u f(u) v The complete algorithm :  The complete algorithm for all u /*increasing mass values from 0 to M/2 */ for all v /*decreasing mass values from M to M/2 */ if (u > f[v]) else if (u < f[v]) If (u,v)E /*maxI is the score of the best interpretation*/ maxI = max {maxI,S[u,v]} De Novo: Second issue:  De Novo: Second issue Given only b,y ions, a forbidden pairs path will solve the problem. However, recall that there are MANY other ion types. Typical length of peptide: 15 Typical # peaks? 50-150? #b/y ions? Most ions are “Other” a ions, neutral losses, isotopic peaks…. De novo: Weighting nodes in Spectrum Graph:  De novo: Weighting nodes in Spectrum Graph Factors determining if the ion is b or y Intensity Support ions Isotopic peaks (InsPecT’) De novo: Weighting nodes:  De novo: Weighting nodes A probabilistic network to model support ions (Pepnovo) De Novo Interpretation Summary:  De Novo Interpretation Summary The main challenge is to separate b/y ions from everything else (weighting nodes), and separating the prefix ions from the suffix ions (Forbidden Pairs). As always, the abstract idea must be supplemented with many details. Noise peaks, incomplete fragmentation In reality, a PRM is first scored on its likelihood of being correct, and the forbidden pair method is applied subsequently.

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