Proteomics Informatics Protein characterization I: - PowerPoint PPT Presentation

Proteomics Informatics – Protein characterization I: post-translational modifications (Week 10)

Post-translational modification • Biologically important post-translational modification (phosphorylation, acetylation, glycosylation, etc.) • Introduced on purpose during sample preparation (alkylation, iTRAQ, TMT etc.) • Side-products of sample preparation (oxidation, deamidation, carbamylation, formylation etc.)

Post-translational modification Mann and Jensen, Nature Biotech. 21, 255 (2003)

Phosphorylation examples Unmodified pS18 pT5 b y b y b y" 1 F 1 F 1 F --- --- --- --- --- --- 2 I 2 I 2 I 261.1556 2163.024 261.1556 2243.024 261.1556 2243.024 3 C 3 C 3 C 421.1862 2049.94 421.1862 2129.94 421.1862 2129.94 4 V 4 V 4 V 520.2546 1889.909 520.2546 1969.909 520.2546 1969.909 5 T 5 T 5 T 621.3022 1790.841 621.3022 1870.841 701.3022 1870.841 6 P 6 P 6 P 718.3549 1689.793 718.3549 1769.793 798.3549 1689.793 7 T 7 T 7 T 819.4025 1592.741 819.4025 1672.741 899.4025 1592.741 8 T 8 T 8 T 920.4502 1491.693 920.4502 1571.693 1000.45 1491.693 9 C 9 C 9 C 1080.481 1390.645 1080.481 1470.645 1160.481 1390.645 10 S 10 S 10 S 1167.513 1230.615 1167.513 1310.615 1247.513 1230.615 11 N 11 N 11 N 1281.556 1143.583 1281.556 1223.583 1361.556 1143.583 12 T 12 T 12 T 1382.603 1029.54 1382.603 1109.54 1462.603 1029.54 13 I 13 I 13 I 1495.687 928.4923 1495.687 1008.492 1575.687 928.4923 14 D 14 D 14 D 1610.714 815.4083 1610.714 895.4083 1690.714 815.4083 15 L 15 L 15 L 1723.798 700.3814 1723.798 780.3814 1803.798 700.3814 16 P 16 P 16 P 1820.851 587.2974 1820.851 667.2974 1900.851 587.2974 17 M 17 M 17 M 1951.891 490.2447 1951.891 570.2446 2031.891 490.2447 18 S 18 S 18 S 2038.923 359.2042 2118.923 439.2042 2118.923 359.2042 19 P 19 P 19 P 2135.976 272.1722 2215.976 272.1722 2215.976 272.1722 20 R 20 R 20 R --- 175.1195 --- 175.1195 --- 175.1195

Potential modifications

Enrichment Strategies for the Detection of Phosphorylated Peptides

Enrichment Strategies for the Detection of Phosphorylated Peptides Unphosphorylated single phosphorylation multiple phosphorylation • Hydrophilic Interaction Chromatography (HILIC) • Phosphopeptides elute later than their unphosphorylated counterparts • Stationary phase is hydrophilic • Mobile phase is hydrophobic

Enrichment Strategies for the Detection of Phosphorylated Peptides SCX Time (min) neutral peptides basic peptides • Strong Cation Exchange Chromatography • Stationary phase is negatively charged • Mobile phase is a buffer that is increasing the pH (if peptide becomes neutral it elutes) • Neutral peptides elute earlier: XXpSxxxxxR/K • Positive peptides elute late: XXXXHXXXXR/K

Several Strategies are often combined

Loss of the phosphate group

Localization of modifications 1.2 1 Probability of Localization 0.8 Phosphopeptide identification 0.6 0.4 0.2 0 0 5 10 15 20 25 Number of fragment ions m precursor = 2000 Da ∆ m precursor = 1 Da ∆ m fragment = 0.5 Da Phosphorylation

Localization of modifications 1.2 Probability of Localization 1 d min >=3 for 47% 0.8 Localization (d min =3) of human tryptic peptides 0.6 0.4 0.2 ID 3 0 0 5 10 15 20 25 Number of fragment ions m precursor = 2000 Da ∆ m precursor = 1 Da ∆ m fragment = 0.5 Da Phosphorylation

Localization of modifications 1.2 Probability of Localization 1 d min =2 for 33% of 0.8 human tryptic Localization (d min =2) peptides 0.6 0.4 ID 0.2 3 2 0 0 5 10 15 20 25 Number of fragment ions m precursor = 2000 Da ∆ m precursor = 1 Da ∆ m fragment = 0.5 Da Phosphorylation

Localization of modifications 1.2 Probability of Localization 1 d min =1 for 20% of 0.8 human tryptic peptides 0.6 Localization (d min =1) 0.4 ID 3 0.2 2 1 0 0 5 10 15 20 25 Number of fragment ions m precursor = 2000 Da ∆ m precursor = 1 Da ∆ m fragment = 0.5 Da Phosphorylation

Localization of modifications 1.2 Probability of Localization 1 0.8 0.6 0.4 ID Localization 3 2 0.2 (d=1*) 1 1* 0 0 5 10 15 20 25 Number of fragment ions m precursor = 2000 Da ∆ m precursor = 1 Da ∆ m fragment = 0.5 Da Phosphorylation

Localization of modifications Peptide with two possible modification sites

Localization of modifications Peptide with two possible modification sites MS/MS spectrum Intensity m/z

Localization of modifications Peptide with two possible modification sites Matching MS/MS spectrum Intensity m/z

Localization of modifications Peptide with two possible modification sites Matching MS/MS spectrum Intensity m/z Which assignment does the data support? 1, 1 or 2, or 1 and 2?

Visualization of evidence for localization AAYYQK AAYYQK

Visualization of evidence for localization AAYYQK AAYYQK

Visualization of evidence for localization 1 2 3 1 2 3

Estimation of global false localization rate using decoy sites By counting how many times the phosphorylation is localized to amino acids that can not be phosphorylated we can estimate the false localization rate as a function of amino acid frequency. 0.02 0.02 False localization frequency 0.015 0.015 0.01 0.01 0.005 0.005 0 0 Y 0 0 0.05 0.05 0.1 0.1 0.15 0.15 Amino acid frequency

How much can we trust a single localization assignment? If we can generate the distribution of scores for assignment 1 when 2 is the correct assignment, it is possible to estimate the probability of obtaining a certain score by chance for a given peptide sequence and MS/MS spectrum assignment. 1. 2. S m m m 1 > S S F S dS 1 ( ) 2 2 2 2 ∫ p 1 1 2 = 0 ∞ S m F dS ( ) S 1 2 2 2 ∫ 1 1 1 0 2 S 1

Is it a mixture or not? If we can generate the distribution of scores for assignment 2 when 1 is the correct assignment, it is possible to estimate the probability of obtaining a certain score by chance for a given peptide sequence and MS/MS spectrum assignment. 1. 2. S m m m 1 > S S 2 1 1 1 2 ∫ F S dS ( ) 2 2 1 = 0 p m ∞ S 2 1 1 1 ∫ 2 F S dS ( ) 2 2 0 1 S 2

Localization of modifications Peptide with two possible modification sites Matching MS/MS spectrum Intensity m/z Which assignment does the data support? 1, 1 or 2, or 1 and 2? p p p p 2 1 th and ≤ ≤ ⇒ 1 and 2 th 1 2 p p p p 2 th and 1 ≤ > ⇒ 1 th 1 2 p p p p p p ≤ S S m m 2 1 2 1 th and ( ) > ≤ ⇒ ≥ ⇒ Ø th 1 2 1 2 1 2 p p p p 2 1 th and > > ⇒ 1 or 2 th 1 2

Top down / bottom up Top down Bottom up intensity mass/charge

Charge distribution Top down Bottom up 2+ 27+ intensity intensity 31+ 3+ 4+ 1+ mass/charge mass/charge

Isotope distribution Top down Bottom up m= 1878 Da intensity intensity mass/charge mass/charge

Fragmentation Top down Bottom up Fragm gmenta tati tion on

Alternative Splicing Top down Exon 1 2 3 Bottom up

Correlations between modifications Top down Bottom up

The Nucleosome Core Complex H3 H3 ‘tail’ H4 H2A H2B Luger et al., Nature, 389, 251-260, 1997

The N-terminal Tails of Histone H3 and H4 Ac M M M P M M Ac M Ac P M M P P M P H3 1-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPTVALRE-50 Ac Ac Ac Ac Ac Ac M M P H4 1-SGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYE-52 P Phosphorylation M Methylation: mono-, di-, or trimethylation Ac Acetylation

The Histone Code Hypothesis Specific post translational modifications (PTMs) of the N-terminal tails of histones function as a scaffold for binding of protein factors leading to transcriptional activation or inactivation. Jenuwein, T., Allis, C.D., Science, 293, 2001

Interdependence of Modifications is lost in Standard Mass Spectrometry Analysis Ac M Ac M P Ac M M M M M P M P P M Ac Ac P H3 1-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPTVALRE-50 M TKQTAR 3-8 M P Ac KSTGGKAPR 9-17 M Ac KQLATKAAR 18-26 M Ac KSAPATGGVKKPHR 27-40 41-50 YRPTVALRE

Histone Proteins are a Highly Complex Mixture of a Single Protein…. M M M M ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE M Ac ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE M M M M ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE M Ac ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE M M M M ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE M M M M ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE ……………… and many many more!

Protocol LTQ-ETD/PTR LTQ-FTMS Glu-C generated N-terminal H3 peptide (1-50) N 50 4 9 14 18 23 27 36 37 546.3 547.6 +10 +11 549.1 • Isolate m/z ± 0.5 Da +9 550.4 +12 551.9 +8 +7 • 60 ms ETD 544.9 m/z •~ 3 min acquisition 245.2 + 10 charge states 346.3 982.5 502.4 ∆ 1.4 Da ∆ 1.4 Da 824.5 ∆ 1.4 Da 892.5 630.5 731.5 672.3 1647.9 1055.6 288.1 571.3 802.5 479.9 958.6 1715.0 401.8 1216.7 1784.1 1129.6 1515.4 1878.2 1255.2 1373.8 1424.8 1937.8 1616.0 m/z m/z