thermo fisher scientific visit - microsoft · detailed manual analysis (uses .puf files) drill ......

PROSIGHT INTRODUCTION Neil Kelleher

OVERVIEW

History and terms

How ProSight Handles Variability

Creating DBs

Search Logic (Modes)

Scoring

Targeted Workflow

HT Workflow

New Stuff !!

ProSightPC v1.0 (released Fall, 2006)

ProSightPC 1.0, 2.0, 3.0 (Developed Since 2004)

WHAT DOES PROSIGHT DO?

Requires:

Input database

MS Data

Searches data against input database

Provides Results

Input Data(Sequence+ MS Data)

Searching Results

400 600 800 1000 1200 1400m/z

THE CANONICAL TOP DOWN EXPERIMENT

Intact MS1 216K RP

500 ms

MS2 108K RP

275 ms Top 3 (transient, reaction time, overhead)

1.5 s Cycle Time

700 800 900 1000 1100 1200m/z

Data Dependent MS/MS

ProSight SearchIntact Mass

DeterminationFragment Mass

Determination

MS1 Injection

~1 msMS2 Injection

~50 ms

How Most Search Engines Handle

Variation

AATASAFGSK (Pot. Phospho on T3, S5, & S9)

AATASAFGSK

AATASAFGSK

AATASAFGSK AATASAFGSK

AATASAFGSK


AATASAFGSK

AATASAFGSK


AATASAFGSK


T3

S5

S9

Unmod S9 S5 S5,S9 T3 T3,S9 T3,S5 T3,S5,S9

How ProSightPC/PD Handles Protein

Variation

• 4 Proteoforms

– PDCD5

– PDCD5 + K63Ac

– PDCD5 + S119Ph

– PDCD5 + K63Ac +

S119Ph

• 262,144 Proteoforms

in Typical Search

Engine using

variable

modifications

Sources of

variability• Alternative Splices

• Endogenous

Cleavages

• Other large sequence

changes

Isoform: Large changes to amino acid sequence

Protein Isoform

Proteoform: The complete set of modifications on an amino acid sequence

Proteoform (Observed)

What is a Proteoform, Exactly?

The Proteoform:

A distinct molecular form of a protein product arising from a single gene

-- The Consortium for Top DownProteomics

CannonicalSequence(UniProtKB)

Endogenous proteolysis mRNA splicingMutationsSNPs

Site specific features-govern activity, localization, interactions, half-life

Smith, L.S., Kelleher, N.L., & The Consortium for Top Down Proteomics, Nat. Methods, 2013, 10, 186–187.

Proteoform: A Single Term to Capture Protein 1o Structure

Smith, L.S., Kelleher, N.L., & The Consortium for Top Down Proteomics, Nat. Methods, 2013, 10, 186–187.

Swiss-Prot Entry for HMGA1

ID HMGA1_HUMAN Reviewed; 107 AA.

AC P17096; P10910; Q5T6U9; Q9UKB0;

DT 01-AUG-1990, integrated into UniProtKB/Swiss-Prot.

DT 23-JAN-2007, sequence version 3.

DT 05-APR-2011, entry version 140.

DE RecName: Full=High mobility group protein HMG-I/HMG-Y;

DE Short=HMG-I(Y);

DE AltName: Full=High mobility group AT-hook protein 1;

DE Short=High mobility group protein A1;

DE AltName: Full=High mobility group protein R;

GN Name=HMGA1; Synonyms=HMGIY;

OS Homo sapiens (Human).

OX NCBI_TaxID=9606;

FT INIT_MET 1 1 Removed.

FT CHAIN 2 107 High mobility group protein HMG-I/HMG-Y.

FT MOD_RES 2 2 N-acetylserine (By similarity).

FT MOD_RES 15 15 N6-acetyllysine.

FT MOD_RES 26 26 Asymmetric dimethylarginine; in isoform HMG-I alternate.

FT MOD_RES 26 26 Omega-N-methylarginine; in isoform HMG-I alternate.

FT MOD_RES 26 26 Symmetric dimethylarginine; in isoform HMG-I; alternate.

FT MOD_RES 36 36 Phosphoserine.

FT MOD_RES 39 39 Phosphothreonine.



FT MOD_RES 53 53 Phosphothreonine.

FT MOD_RES 58 58 Asymmetric dimethylarginine; by PRMT6; alternate.

FT MOD_RES 58 58 Omega-N-methylarginine; by PRMT6; alternate.

FT MOD_RES 60 60 Asymmetric dimethylarginine; by PRMT6;

FT MOD_RES 60 60 Omega-N-methylarginine; by PRMT6;

FT MOD_RES 99 99 Phosphoserine; in isoform HMG-I and isoform HMG-Y.

FT MOD_RES 102 102 Phosphoserine; by CK; in isoform HMG-I and isoform HMG-Y.

FT MOD_RES 103 103 Phosphoserine; by CK; in isoform HMG-I and isoform HMG-Y.

FT VAR_SEQ 35 45 Missing (in isoform HMG-Y).

FT VAR_SEQ 66 107 NKGAAKTRKTTTTPGRKPRGRPKKLEKEEEEGISQESSEEE

FT Q -> KNWRRRKRRASRRSPRRRSSDPCVPPAPHWRSSFLL

FT GLDSFAPLPPPPPLPQAHHHHRLWPPPPSSTCALTTTLHST

FT PAAAGLPWAEWGAVFPWPQFPAPPAHPRIHTCPPGQG (in

FT isoform HMG-R).

SQ SEQUENCE 107 AA; 11676 MW; E9C4E3F2200914B8 CRC64;

MSESSSKSSQ PLASKQEKDG TEKRGRGRPR KQPPVSPGTA LVGSQKEPSE VPTPKRPRGR

VAR_SEQ indicates splice variants

MOD_RES indicates post-translational

modifications

This entry will

produce 3 ProSight

Base Sequences and

32,256 ProSight

Protein Forms

THE VALUE OF PLACING PTMS IN PROTEIN DATABASES (AND SEARCHING FOR THEM)

N CN-terminal acetylation is not in database

Observed Fragment Ions

Ac

N-terminal

acetylation,

+42 Da

Absolute Mass Values of N-terminal ions

Do NOT match

C

N-terminal acetylation is in database

Absolute Mass Values of N-terminal ions

Do match

Ac

Ac

Ac

Correct Gene Family

Correct Gene

Correct Protein Isoform

Shotgun Annotation Extended to the Whole Human Proteome

Roth, Kelleher, and Team, Mol Cell Proteomics, 2005, 7, 1002-1008.

HUMAN PROTEIN DATABASES FOR MASS SPECTRAL SEARCHING

UniProt entries for Homo

sapiens, Release 2011-04

Features Recognized

Reduce Redundancy at the Gene and Protein

Level

Allow 213

PTMs/SNPs possible per

sequence

50,190 Unique

Base Sequences

20225 Entries

8.5 Million

Isoforms

(5 GB)

• Initial Methionine

• Peptide Cleavage

Events

• Known Modifications

• Sequence Variants

• Conflicts

The Process of “Shotgun Annotation”Allows automated detection of combinatorial

modifications

DATABASE MANAGER

DATABASE CREATION

TWO GENERAL STRATEGIES TO IDENTIFY PEPTIDES TO MAP TO PROTEINS

24

MS1 only: match intact mass of peptide ion to predicted peptides

peptide ion

accurate mass of peptide ion

Peptide sequencing: fragment peptides, gain sequence information

fragmentation spectrum of peptide

Intact mass & sequence tag of GWNPYYK

MVVSIGWNPYYK

+ +

Search database

300 1500m/z

300 1500m/z

728.868z=2

SEARCH MODES (THERE ARE 5 IN PROSIGHTPC)

Absolute Mass:

Interrogates all protein forms within a window of

the observed intact mass.

Biomarker:

Searches all possible sub-sequences to find

those with the observed intact mass.

DELTA M MODEError tolerant search: A delta mass is placed at both termini and fragment matching is performed.

∆M = observed precursor mass – candidate mass

N C

N C

N C

∆M

∆M

Match both directions

Match towards N terminus

Match towards C terminus

ProSightPC

Candidate

Warehouses

Detailed Manual

Analysis

(uses .PUF files)

Drill

Down

ProSightPD 1.0 Nodes

ProSight Integration into PD 2.x

BETTER GRAPHICAL SUPPORT IN PROSIGHTPD(PROTEOME DISCOVERER NEEDS TO SUPPORT TOP DOWN BETTER!)

AX – green, angled flag

BY – blue, angled flag

CZ* – red, square flag

If both AB or XY, displays as

BY

TOP DOWN DATA TYPES

Lo/Hi – Respect in ProSightPD

Med/Hi – Respect in ProSightPD

Hi/Hi – cRAWler in ProSightPD and

ProSightPC 3.0

Precursor Data Types

Low Resolution = “Lo”

MA: ~1 - 20 Da

Medium Resolution =

“Med”

MA: 1 – 0.1 Da

High Resolution = “Hi”

MA: 1 – 20 ppm

• Ion Trap (e.g.,

LTQ)

• (Quadrupole)

• Time-of-Flight (TOF)

• Magnetic Sector

• Short Transient

Orbitrap (RP ~6000)

• Orbitrap

APPLICATION OF THE RESPECT ALGORITHM TO IT-FT (LO-HI) DATA FROM PSEUDOMONAS AERUGINOSA

(PROVIDED BY IOANNA NTAI, PCE , NORTHWESTERN U.)

JANUARY, 24TH 2014

CREATING SEARCHES

SCORING MODEL

Statistical Model based on the Poisson Distribution• If an event is expected to occur times, then the probability of it occurring k times is

given by the Poisson distribution:

• If f is the total number of observed fragments, and n is the number of matching fragments, we may estimate the probability of a random protein match given f and n:

where x is the mean probability that a mass of an observed fragment ion will randomly match one from a generic protein.

!

,k

ekP

k

221.111

1

!),(

a

xfn

Mx

n

exffnP

[Eq.1]

[Eq.2]

SCORING MODEL

Given the probability of a random protein match with a particular n, f, and Ma, we can now estimate the probability of getting a match at least as good as we have by random chance:

where p is the p-value assuming our Poisson model – what we call the P-Score.

The Expectation Value (e-value) reported in ProSightPC is simply the P-Score scaled to the size of the search space.

1

0 !1

n

i

xfn

n

exfp [Eq.3]

CRAWLER ALGORITHM

Creates MS/MS Experiments during a high throughput run

Implementation

Groups fragment scans based on their isolation m/z and retention times

For each fragment scan group

Averages fragment scans

Runs Xtract/THRASH to create neutral fragment masses

Averages precursor scans based on proximity

Runs Xtract/THRASH in isolation window to create neutral precursor masses

PROSIGHT HT: ITERATIVE SEARCHING

High-throughput data needs to be searched in a high-throughput fashion

Minimize user intervention, minimize analysis time, maximize quality of hits

Iterative search logic automates user intuition regarding cost/benefit of searches and quality of results

Binary search tree with decisions driven by search results

PROSIGHT HT: ITERATIVE SEARCHING

• Fast, Narrow, Specific Searches on Small Databases

• Contaminant Filtering (Bottom-Up/Middle-Down)

• Targeted Protein Identification/Characterization

• Narrow Absolute Mass Searches on Large Databases

• Proteomic Investigations

• Wide Absolute Mass Searches on Small Databases

• Characterization of Unknown cSNPs and PTMs on

Targeted Proteins

• Wide Absolute Mass Searches on Large Databases

• Proteomic Characterization of Unknown cSNPs and PTMs

• Narrow Biomarker Searches

• Identification of Unknown Proteolytic Products

• Wide Biomarker Searches

• Identification of Unknown Proteolytic Products with Unknown

cSNPs and PTMs

1

2

3

4

Searc

h C

om

ple

xity

Se

arc

h S

pe

ed

Load

Load

Load

Load Load

HIGH THROUGHPUT WIZARD

HIGH THROUGHPUT LOGIC

HIGH THROUGHPUT RESULTS

SIMPLIFIED SEARCH RESULTS

Top Down Data Types

Lo/Hi – Respect in ProSightPD

Mid/Hi – Respect in ProSightPD

(Express Scans for MS1)

High/High – cRAWler in

ProSightPD and ProSightPC 3.0

Precursor Data Types

Low Resolution = “Lo”

MA: ~1 - 20 Da

Medium Resolution =

“Med”

MA: 1 – 0.1 Da

High Resolution = “Hi”

MA: 1 – 20 ppm

• Ion Trap (e.g.,

LTQ)

• (Quadrupole)

• Time-of-Flight (TOF)

• Magnetic Sector

• Short Transient

Orbitrap (RP ~6000)

• Orbitrap

The “C-Score”: Improving Proteoform Characterization

UVPD Now fully

Supported in

ProSightPC 4.0

and

ProSightPD 1.x

UVPD meets Top Down meets ProSight

With Jenny Brodbelt and San Jose/Bremen

R & E/D

*Data from a modified Orbitrap Elite

Fragment Map Comparison

UVPD of RL29_ECOBW, 50S

ribosomal protein L29

Ions Searched

A, A+, X, X+

B, Y, Y-

C, Z*

Better Graphical Support in ProSightPD

(Proteome Discoverer needs to support

top down better!)

AX – green, angled flag

BY – blue, angled flag

CZ* – red, square flag

If both AB or XY, displays

as BY

thermo fisher scientific visit - microsoft · detailed manual analysis (uses .puf files) drill ......

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