Sequence Based Analysis Tutorial NIH Proteomics Workshop Lai-Su Yeh, Ph.D. Protein Information Resource at Georgetown University Medical Center

2 Retrieval, Sequence Search & Classification Methods Retrieve protein info by text / UID Sequence Similarity Search BLAST, FASTA, Dynamic Programming Family Classification Patterns, Profiles, Hidden Markov Models, Sequence Alignments, Neural Networks Integrated Search and Classification System

3 Sequence Similarity Search (I) Based on Pair-Wise Comparisons Dynamic Programming Algorithms Global Similarity: Needleman-Wunch Local Similarity: Smith-Waterman Heuristic Algorithms FASTA: Based on K-Tuples (2-Amino Acid) BLAST: Triples of Conserved Amino Acids Gapped-BLAST: Allow Gaps in Segment Pairs PHI-BLAST: Pattern-Hit Initiated Search PSI-BLAST: Position-Specific Iterated Search

4 Sequence Similarity Search (II) Similarity Search Parameters Scoring Matrices – Based on Conserved Amino Acid Substitution Dayhoff Mutation Matrix, e.g., PAM250 (~20% Identity) Henikoff Matrix from Ungapped Alignments, e.g., BLOSUM 62 Gap Penalty Search Time Comparisons Smith-Waterman: 10 Min FASTA: 2 Min BLAST: 20 Sec

5 Feature Representation Features of Amino Acids: Physicochemical Properties, Context (Local & Global) Features, Evolutionary Features Alternative Amino Acids: Classification of Amino Acids To Capture Different Features of Amino Acid Residues

6 Substitution Matrix Likelihood of One Amino Acid Mutated into Another Over Evolutionary Time Negative Score: Unlikely to Happen (e.g., Gly/Trp, -7) Positive Score: Conservative Substitution (e.g., Lys/Arg, +3) High Score for Identical Matches: Rare Amino Acids (e.g., Trp, Cys)

7 Secondary Structure Features  Helix Patterns of Hydrophobic Residue Conservation Showing I, I+3, I+4, I+7 Pattern Are Highly Indicative of an  Helix (Amphipathic)  Strands That Are Half Buried in the Protein Core Will Tend to Have Hydrophobic Residues at Positions I, I+2, I+4, I+6

8 BLAST BLAST (Basic Local Alignment Search Tool) Extremely fast Robust Most frequently used It finds very short segment pairs (“seeds”) between the query and the database sequence These seeds are then extended in both directions until the maximum possible score for extensions of this particular seed is reached

9 BLAST Search From BLAST Search Interface Table-Format Result with BLAST Output and SSEARCH (Smith- Waterman) Pair-Wise Alignment Link to NCBI taxonomy Click to see alignment Links to iProClass and UniProtKB reports Link to PIRSF report Click to see SSearch alignment

10 Blast Result & Pairwise Alignment BLAST Aligment

11 How do you build a tree? Pick sequences to align Align them Verify the alignment Keep the parts that are aligned correctly Build and evaluate a phylogenetic tree Integrated Analysis

12 Pairwise alignment: Calculate distance matrix Mean number of differences per residue Unrooted Neighbor-Joining Tree Branch length drawn to scale Rooted NJ Tree (guide tree) Root place at a position where the means of the branch lengths on either side of the root are equal Progressive Alignment guided by the tree Alignment starts from the tips of the tree towards the root Thompson et al., NAR 22, 4675 (1994). Multiple Sequence Alignment: CLUSTALW

13 PIR Multiple Alignment and Tree From Text/Sequence Search Result or CLUSTAL W Alignment Interface

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15 PIR Pattern Search From Text/Sequence Search Result or Pattern Search Interface P-[IV]-[WY]-x(3)-H-[MR]-V-x(3,4)-Q-x(1,2)-D-x(4,5)-G-A-N Alignment of a region involved in catalytic activity Create Pattern and search in database: A B O05689 Test sequence against PROSITE database Signature Patterns for Functional Motifs

16 Pattern Search Result (I) A.One Query Pattern Against UniProtKB or UniRef100 DBs Display the query pattern Links to iProClass and UniProtKB reports Link to NCBI taxonomy Link to PIRSF report Indicate pattern sequence region(s)

17 Pattern Search Result (II) B.One Query Sequence Against PROSITE Pattern Database

18 Profile Method Profile: A Table of Scores to Express Family Consensus Derived from Multiple Sequence Alignments Num of Rows = Num of Aligned Positions Each row contains a score for the alignment with each possible residue. Profile Searching Summation of Scores for Each Amino Acid Residue along Query Sequence Higher Match Values at Conserved Positions

19 Prosite PS50157 profile for Zinc finger C2H2

20 Search One Query Protein Against all the Full-length and Domain HMM models for the fully curated PIRSFs by HMMER The matched regions and statistics will be displayed. Shows PIRSF that the query belongs to Statistical data for all domains Statistical data per domain Alignment with consensus sequence 1 PIRSF scan

21 Lab Section

22 Rat eye lens phosphoproteomics in normal and cataract Kamei et al., Biol. Pharm. Bull., Normal Cataract (-)pI(+) Mw More phosphorylated spots in cataract sample. Digestion and MS from Spot 16 gave these peptides: MDVTIQHPWFKR ALGPFYPSR CSLSADGMLTFSG YRLPSNVDQSALS We want to identify the protein(s) that contain these peptides Use Peptide Search MDVTIQHPWFKR

23 Peptide Search

24 Links to iProClass and UniProtKB reports Link to NCBI taxonomy Link to PIRSF report Matching peptide highlighted in the sequence Sorting arrows Peptide Search & Results Species restricted search Search in UniProtKB, 23 proteins

25 Batch Retrieval Results (I) Retrieve more sequences Retrieve multiple proteins in from iProClass using a specific identifier or a combination of them Provides a means to easily retrieve and analyze proteins when the identifiers come from different databases

26 ID Mapping

27 Blast Similarity Search >P24623 Perform sequence similarity search What proteins are related to rat CRYAA?

28 Blast Search Results BLAST (partial) result for CRYAA_RAT in UniProtKB database BLAST alignment with the human protein

29 Pairwise Alignment

30 UniProtKBDatabase and unique UniParc sequences PIR protein family classification database PIR Text Search ( ( Let’s search for human crystallins

31 Refine your search or start over Display PDB ID Let’s look for crystallins which have 3D structure

32 Domain Display allows to compare simultaneously Pfam domains present in multiple proteins Let’s perform a multiple alignment on the sequences containing PF00030 Share same domain architecture

33 Multiple Alignment

34 Interactive Phylogenetic Tree and Alignment Beta B1 and gamma crystallins share the same domains, SCOP fold and share significant sequence similarity suggesting that they are related

35 Pattern Search (I) Search for proteins containing this pattern (PS00225) in rat Select P07320 and perform a pattern search

36 Pattern Search Result Beta and gamma Crystallins have multiple copies of this pattern