Validating sequence sequence assignments for peptide assignments for peptide fragmentation patternsfragmentation patterns

by Karen Jonscherby Karen Jonscher

www.proteomesoftware.comwww.proteomesoftware.com503-244-6027503-244-6027

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With the advent of high throughput proteomics, data is being generated at an astonishing rate.

It has become clear that validating peptide sequence assignments generated by database search engines is an increasingly important, but often overlooked aspect of protein identification using tandem mass spectrometry.

In this tutorial you will learn about some of the factors important in low energy peptide fragmentation and how to use this information to accept or reject database search engine peptide sequence assignments.

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Mascot Score = 101Mascot Score = 101

Mascot Score = 102Mascot Score = 102

Why is validating data important?Why is validating data important?

• Every database search, for a variety of reasons, generates false positive and false negative assignments. We would like to at least reduce, if not eliminate, these incorrect hits.• Decisions, often involving a great deal of money for bioassays, will be made downstream of our peptide identification. In this era of tight funding, it is crucial that the data upon which these crucial decisions rest are completely accurate.

High quality spectrum High quality spectrum with good signal-to-with good signal-to-noise and all of the noise and all of the dominant ions dominant ions assignedassigned..

Poor quality spectrum Poor quality spectrum with low signal-to-with low signal-to-noise. Fragment ions noise. Fragment ions are most likely are most likely randomly assigned to randomly assigned to noise peaks.noise peaks.

These two fragment ion spectra from a multi-dimensional LC proteomics experiment using a quadrupole ion trap were searched with Mascot. They both had similar scores. Are these two spectra equivalent?

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How do peptides break apart?How do peptides break apart?

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In order to assess whether a sequence assignment is correct or not, it is important to understand how and why peptides break apart.

Under low energy dissociation conditions, peptides primarily fragment at the C – N bond. If the charge is retained on the N-terminal end of the peptide, the ion is known as a b-type ion. If the charge is retained on the C-terminal end, the ion is termed a y-type ion. The fragmentation energy in some instruments, especially triple quadrupoles or quadrupole-time-of-flight hybrids (Q-TOFs) is often sufficient to generate cleavage at the C-C bond as well, causing loss of CO from the b ion. These ions are known as a-type ions.

HH22N-AN-A++ = b = b11

HH22N-ABN-AB++ = b = b22

HH22N-ABCN-ABC++ = b = b33

HH22N-ABCDN-ABCD++ = b = b44

HH22N-ABCDEN-ABCDE++ = b = b55

HH22N-ABCDEFN-ABCDEF++ = b = b66

HH22N-ABCDEFGN-ABCDEFG++ = b = b77

yy11 = = ++G-COOHG-COOH

yy22 = = ++FG-COOHFG-COOH

yy33 = = ++EFG-COOHEFG-COOH

yy44 = = ++DEFG-COOHDEFG-COOH

yy55 = = ++CDEFG-COOHCDEFG-COOH

yy66 = = ++BCDEFG-COOHBCDEFG-COOH

yy77 = = ++ABCDEFG-COOHABCDEFG-COOH

Peptides dissociate into Peptides dissociate into nested sets of fragmentsnested sets of fragments

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AA BB CC DD EE FF GGHH22NN COOHCOOH-- --

bb11 bb22 bb33 bb44 bb55 bb66

yy66 yy55 yy44 yy33 yy22 yy11

If A, B, C, D, E, F and G represent different amino acids, this peptide can dissociate to form the following fragments:

Rel

ati

ve

Ab

un

da

nce

m/z

AB

b2

C

b3

D

b4

E

b5

Fb6

G

FG

y2

E

y3

D

y4

C

y5

B

y6

A

The peptide amino acid sequence can be deduced by calculating the difference in mass between peaks. If the mass corresponds to an amino acid residue (a table is shown in the next slide) then that amino acid is assigned to the peak representing the difference. The largest y-type ion will appear anywhere between 57 to 186 amu below the mass of the precursor ion. The smallest y ion will appear at the amino acid residue mass plus 19 amu. The largest b ion will be at 18 amu plus the residue mass below the precursor and the smallest b ion will be at the residue mass + 1. Using this information, we can interpret fragmentation spectra to deduce the amino acid sequence. Keep clicking to view an ion series.

Mass difference reflects Mass difference reflects peptide sequencepeptide sequence

AA BB CC DD EE FF GGHH22NN COOHCOOH-- --

bb11 bb22 bb33 bb44 bb55 bb66

yy66 yy55 yy44 yy33 yy22 yy11

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Name Symbol Mass (-H2O) Side Chain Immonium/Related Ions

Alanine A, Ala 71.079 CH3- 44

Arginine R, Arg 156.188 HN=C(NH2)-NH-(CH2)3- 129/59, 70. 73, 87, 100, 112

Asparagine N, Asn 114.104 H2N-CO-CH2- 87/70

Aspartic acid D, Asp 115.089 HOOC-CH2- 88/70

Cysteine C, Cys 103.145 HS-CH2- 76

Glutamine Q, Gln 128.131 H2N-CO-(CH2)2- 101

Glutamic acid E, Glu 129.116 HOOC-(CH2)2- pro102/56, 84, 129

Glycine G, Gly 57.052 H- 30

Histidine H, His 137.141N=CH-NH-CH=C-CH2- |

__________|110/82, 121, 123, 138, 166

Isoleucine I, Ile 113.160 CH3-CH2-CH(CH3)- 86/44, 72

Leucine L, Leu 113.160 (CH3)2-CH-CH2- 86/44, 72

Lysine K, Lys 128.17 H2N-(CH2)4- 101/70, 84, 112, 129

Methionine M, Met 131.199 CH3-S-(CH2)2- 104/61

Phenylalanine F, Phe 147.177 Phenyl-CH2- 120/91

Proline P, Pro 97.117 -N-(CH2)3-CH- |_________| 70

Serine S, Ser 87.078 HO-CH2- 60

Threonine T, Thr 101.105 CH3-CH(OH)- 74

Tryptophan W, Trp 186.213Phenyl-NH-CH=C-CH2- |

___________|159/77, 117, 130, 123, 170, 171

Tyrosine Y, Tyr 163.176 4-OH-Phenyl-CH2- 136/91, 107

Valine V, Val 99.133 CH3-CH(CH2)- 72/41, 55, 69

basic

basic

basic

acidic

acidic

lose water

lose water

suppress b

suppress b

typically dominant

lose ammonia

lose ammonia

lose ammonia

lose ammonia

-H2S=34

-CH3SH=48

Not usually observedin ion traps due to low masscutoff. Same with b1/y1.

abundant y

isobaric

isobaric

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The table of common amino acids provides molecular weights for the residues, structures of the side chains, and masses for the low mass immonium ions that result from side chain loss.

These amino acids have chemical properties that need to be considered when validating sequence assignments.

Other things to keep in mindOther things to keep in mind

G-G = 114 = NG-A = 128 = K/QV-G = 156 = RG-E = 186 = WA-D = 186 = WS-V = 186 = WAcG = 99 = VAcA = 113 = L/IAcS = 129 = EAcN = 156 = R

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Some pairs of amino acids add up Some pairs of amino acids add up to the mass of a different amino to the mass of a different amino acid. The same can happen with acid. The same can happen with acetylated amino acids, a common acetylated amino acids, a common modification. modification.

Basic amino acids can generate doubly-charged ions. Ion signal can be intense for cleavages C-terminal to acidic amino acids. These residues also tend to lose water and cyclize to randomly eject portions of the sequence. Isobaric amino acids cannot be differentiated using low energy fragmentation instruments. Loss of water from threonine is particularly intense if the amino acid is near a terminal end of the peptide. If a peptide is tryptic, y1 will either be lysine at 147 or arginine at 175.

13

37

.5

12

76

.51

23

8.6

12

19

.4

11

39

.4

10

91

.4

99

2.4

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We’re going to manually interpret an MS/MS spectrum generated by a quadrupole ion trap.

The deconvoluted mass of the precursor is 1449.38 (the observed ion was doubly charged).

We’ll start by looking at the dominant peaks that are below the mass of the precursor ion.

We’ll look for possible y ions between 57 and 186 amu below 1450 (the (M+H)+ ion) and possible b ions in a window offset by another 18 amu.

Lxx Val Val Phe K/Q Gly Arg

The first ion is at 1337.5. 1450-1337=113=Lxx, therefore we assign the largest y ion as either leucine or isoleucine.

The next ion is at 1276. 1450-18-1276=156=Arg, therefore we assign the largest b ion as arginine. Since the sample was digested with trypsin, we would expect lysine or arginine as the first y ion.

Next we look at the ion at 1238. Assuming it is a y ion, we note that 1337-1238=99=Val, so we assign the next y ion as Val.

Continuing in this manner, we find b ions at 1276-1219=57=Gly and 1219-1091=128=Lys/Gln and y ions at 1238-1139=99=Val and 1139-992=147=Phe.

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37

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12

76

.512

38.

61

21

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39

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10

91

.4

99

2.4

97

5.49

44

.39

27

.3

86

3.4

21

3.0

23

2.0

31

1.8

36

0.2

44

3.3

45

9.0

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Lxx Val Val Phe K/Q Gly Arg

To verify the high mass y ion assignments, we look for the complimentary low mass b ions.

Since the data is from an ion trap, we will probably not see b1.

Therefore, we start by looking for b2.

Since the largest y ion was either leucine or isoleucine and the next y ion in the series was valine, we look for the complementary ion at 113+1+99=213=b2.

The next b ion will result from addition of another valine, therefore we’d expect a signal at 213+99=312=b3.

When phenylalanine is added, we find the b4 ion at 312+147=459.

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12

76

.512

38.

61

21

9.4

11

39

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10

91

.4

99

2.4

97

5.49

44

.39

27

.3

86

3.4

21

3.0

23

2.0

31

1.8

36

0.2

44

3.3

45

9.0

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To verify the high mass b ion assignments, we look for the complimentary low mass y ions.

Since the data is from an ion trap, we will probably not see y1, however the assignment of arginine makes sense given that the sample was digested using trypsin.

Therefore, we start by looking for y2.

Assuming arginine is y1, we look for a signal resulting from the addition of glycine at 156+1+18+57=232=y2. We add the 19 amu to account for the carboxyl group.

The next complementary y ion should be at 232+128=360=y3 resulting from the addition of either lysine or glutamine. Since the sample was digested using trypsin, it is unlikely that the amino acid is lysine, since we would have expected a cleavage there.

Lxx Val Val Phe K/Q Gly ArgQ

1091-975=1161091-944=147=Phe1091-927=164

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38.

61

21

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11

39

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91

.4

99

2.4

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5.49

44

.39

27

.3

86

3.4

b 2y 2

b 3y 3

44

3.3

b 4

83

0.2

48

9.0

50

7.2

54

3.3

58

8.0

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Lxx Val Val Phe GlyArgGlnGlu PheAsn

We look for the next largest b ion below 1091. There are three choices:

1091-975=1161091-944=147=Phe1091-927=164

Only the signal at 944 corresponds to an amino acid residue mass, therefore the next b ion is Phe.

The next largest y ion will appear below 992 Since 992-863=129=Glu, we assign the next y ion as glutamic acid.

The next dominant peak should correspond to a b ion so we look below 944 and note that 944-830=114=Asn.

Validating our high mass ion assignments, we expect to find b5 resulting from addition of glutamic acid at 459+129=588=b5 and

y4 resulting from addition of phenylalanine, 360+147=507=y4.

1091-975=1161091-944=147=Phe1091-927=164

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12

76

.512

38.

61

21

9.4

11

39

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91

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99

2.4

97

5.49

44

.39

27

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86

3.4

b 2y 2

b 3y 3

44

3.3

b 4

83

0.2

48

9.0

54

3.3

y 4

b 5

75

0.3

70

1.1

621.3

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The next largest y ion will appear below 863. Since 863-750=113=Lxx, we assign the next y ion as leucine/isoleucine.

Lxx Val Val Phe Gly ArgGlnGlu PheAsnLxx

The next dominant peak should correspond to a b ion so we look below 830 and note that 830-701=129=Glu.

Glu

The next y ion will appear below 750. Since 750-621=129=Glu, we assign the next y ion as glutamic acid. However, since this is complementary to the b ion we just assigned, our sequence is complete.

1091-975=1161091-944=147=Phe1091-927=164

b 2y 2

b 3y 3

44

3.3

b 44

89

.0

54

3.3

y 4

b 5

b 5

b 7

b 8

b 9

b 10

b 11

y 5

y 6

y 7

y 8

y 9

y 10

y 11

b

yL V V F E L E N F Q G R

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In this example we have observed a complete series of complementary b and y ions. Purple bars indicate b ions while orange bars are for y ions.

# Rank/Sp Id# (M+H)+ deltCn XCorr Sp Ions Reference Peptide --- -------- -------- -------- ------ ------ ---- ---- --------- ------- 1. 1 / 1 0 1450.7694 0.0000 4.7567 2559.1 20/22 CRB1_HUMAN R.LVVFELENFQGR.R 2. 2 / 2 0 1451.7534 0.0254 4.6357 2541.5 20/22 CRB1_HUMAN R.LVVFELEN*FQGR.R 3. 3 / 2 0 1451.7534 0.0571 4.4851 2541.5 20/22 CRB1_HUMAN R.LVVFELENFQ*GR.R 4. 4 / 3 0 1452.7374 0.2804 3.4230 2036.0 18/22 CRB1_HUMAN R.LVVFELEN*FQ*GR.R 5. 5 / 6 0 1451.7569 0.4038 2.8358 1057.7 15/22 TP3B_HUMAN K.LN*M#VKFLQ*VEGR.G 6. 6 / 5 0 1450.7729 0.4619 2.5595 1426.3 17/22 TP3B_HUMAN K.LN*M#VKFLQVEGR.G 7. 7 / 13 0 1450.6549 0.4654 2.5430 840.0 13/22 NEUM_HUMAN R.TKQ*VEKN*DDDQ*K.I 8. 8 / 13 0 1449.6709 0.4752 2.4964 840.0 13/22 NEUM_HUMAN R.TKQ*VEKNDDDQ*K.I 9. 9 / 15 0 1449.6709 0.4757 2.4941 817.4 13/22 NEUM_HUMAN R.TKQ*VEKN*DDDQK.I 10. 10 / 11 0 1451.7055 0.5042 2.3586 843.6 13/22 ING_HUMAN K.S]VETIKEDM#NVK.F 11. 11 / 16 0 1451.8011 0.5145 2.3093 817.3 14/22 GGT5_HUMAN R.VNVYHHLVETLK.F 12. 12 / 4 0 1451.7494 0.5179 2.2932 1458.5 16/20 DESP_HUMAN R.LTYEIEDEKRR.R

Incorrect Identification

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Sequest tenatively matches each spectrum to 12 peptides. Matches are rated with the Xcorr value (Sequest’s score criterion). Here is a poorly rated match. Although most of the dominant ions were assigned, they were not assigned to b and y ions but to water loss and other ions that are generally less abundant.

# Rank/Sp Id# (M+H)+ deltCn XCorr Sp Ions Reference Peptide --- -------- -------- -------- ------ ------ ---- ---- --------- ------- 1. 1 / 1 0 1450.7694 0.0000 4.7567 2559.1 20/22 CRB1_HUMAN R.LVVFELENFQGR.R 2. 2 / 2 0 1451.7534 0.0254 4.6357 2541.5 20/22 CRB1_HUMAN R.LVVFELEN*FQGR.R 3. 3 / 2 0 1451.7534 0.0571 4.4851 2541.5 20/22 CRB1_HUMAN R.LVVFELENFQ*GR.R 4. 4 / 3 0 1452.7374 0.2804 3.4230 2036.0 18/22 CRB1_HUMAN R.LVVFELEN*FQ*GR.R 5. 5 / 6 0 1451.7569 0.4038 2.8358 1057.7 15/22 TP3B_HUMAN K.LN*M#VKFLQ*VEGR.G 6. 6 / 5 0 1450.7729 0.4619 2.5595 1426.3 17/22 TP3B_HUMAN K.LN*M#VKFLQVEGR.G 7. 7 / 13 0 1450.6549 0.4654 2.5430 840.0 13/22 NEUM_HUMAN R.TKQ*VEKN*DDDQ*K.I 8. 8 / 13 0 1449.6709 0.4752 2.4964 840.0 13/22 NEUM_HUMAN R.TKQ*VEKNDDDQ*K.I 9. 9 / 15 0 1449.6709 0.4757 2.4941 817.4 13/22 NEUM_HUMAN R.TKQ*VEKN*DDDQK.I 10. 10 / 11 0 1451.7055 0.5042 2.3586 843.6 13/22 ING_HUMAN K.S]VETIKEDM#NVK.F 11. 11 / 16 0 1451.8011 0.5145 2.3093 817.3 14/22 GGT5_HUMAN R.VNVYHHLVETLK.F 12. 12 / 4 0 1451.7494 0.5179 2.2932 1458.5 16/20 DESP_HUMAN R.LTYEIEDEKRR.R

Correct Identification

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When we compare the incorrect with the correct identification, we see that all of the dominant ions are assigned to b and y ions, with water losses accounting for many of the lower abundance peaks. The rank is 1 and the Xcorr is high in the Sequest output file for the correct ID.

Bad Spectral QualityBad Spectral Quality

Good Spectral QualityGood Spectral Quality

Good ScoreGood Score Bad ScoreBad Score

Bad IDBad IDGood IDGood ID

? ID? ID

Bad IDBad ID

Bad IDBad ID

Good IDGood ID

Bad IDBad ID

Score vs. Spectral QualityScore vs. Spectral Quality

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As we have seen, a high score may be an indicator that an identification is correct.

However, this does not hold true in all cases. In the next few examples, we will see instances where good scores actually corresponded to bad identifications, and bad scores corresponded to good identifications.

The importance of spectral quality is demonstrated and the case of questionable identification is reviewed.

Bad Spectral QualityBad Spectral Quality

Good Spectral QualityGood Spectral Quality

Good ScoreGood Score Bad ScoreBad Score

Bad IDBad ID

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Example 1Example 1

Proteomics DataProteomics DataMascot SearchMascot Search

1. IPI00001661 Mass: 45425 Total score: 111 Peptides matched: 1 Tax_Id=9606 Regulator of chromosome condensation Check to include this hit in error tolerant search or archive report

  Query   Observed   Mr(expt)   Mr(calc)   Delta  Miss Score  Rank   Peptide

   1     950.77    1899.53    1899.00     0.53    0   111     1    VVQVSAGDSHTAALTDDGR

Great score, nice spectrumGreat score, nice spectrum

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The Mascot score is 111. Usually, scores over 40 or 50 typically generate correct identifications.

The score is well beyond the 95% confidence level and is well separated from the other possibilities, generally a positive indicator of a correct ID.

The mass spectrum has a lot of fragment ions and has good signal to noise. Oftentimes, good quality spectra like this provide good search results.

# b b++ b* b*++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ #

1 100.14 50.57         V             19

2 199.27 100.14         V 1800.88 900.94 1783.85 892.43 1782.87 891.94 18

3 327.40 164.21 310.37 155.69     Q 1701.75 851.38 1684.72 842.86 1683.73 842.37 17

4 426.54 213.77 409.51 205.26     V 1573.62 787.31 1556.59 778.80 1555.60 778.30 16

5 513.61 257.31 496.58 248.80 495.60 248.30 S 1474.48 737.75 1457.45 729.23 1456.47 728.74 15

6 584.69 292.85 567.66 284.34 566.68 283.84 A 1387.41 694.21 1370.38 685.69 1369.39 685.20 14

7 641.75 321.38 624.72 312.86 623.73 312.37 G 1316.33 658.67 1299.30 650.15 1298.31 649.66 13

8 756.83 378.92 739.80 370.41 738.82 369.91 D 1259.28 630.14 1242.24 621.63 1241.26 621.13 12

9 843.91 422.46 826.88 413.94 825.90 413.45 S 1144.19 572.60 1127.16 564.08 1126.17 563.59 11

10 981.05 491.03 964.02 482.52 963.04 482.02 H 1057.11 529.06 1040.08 520.54 1039.09 520.05 10

11 1082.16 541.58 1065.13 533.07 1064.14 532.58 T 919.97 460.49 902.94 451.97 901.95 451.48 9

12 1153.24 577.12 1136.21 568.61 1135.22 568.12 A 818.86 409.94 801.83 401.42 800.85 400.93 8

13 1224.32 612.66 1207.29 604.15 1206.30 603.65 A 747.78 374.40 730.75 365.88 729.77 365.39 7

14 1337.48 669.24 1320.45 660.73 1319.46 660.23 L 676.70 338.86 659.67 330.34 658.69 329.85 6

15 1438.58 719.79 1421.55 711.28 1420.57 710.79 T 563.55 282.28 546.51 273.76 545.53 273.27 5

16 1553.67 777.34 1536.64 768.82 1535.65 768.33 D 462.44 231.72 445.41 223.21 444.42 222.72 4

17 1668.76 834.88 1651.73 826.37 1650.74 825.88 D 347.35 174.18 330.32 165.66 329.34 165.17 3

18 1725.81 863.41 1708.78 854.89 1707.79 854.40 G 232.26 116.64 215.23 108.12     2

19             R 175.21 88.11 158.18 79.59     1

The masses of the expected sequence ions are summarized in the data table. Masses labeled in red were observed in the experiment. As we can see, we have a fairly long run of contiguous sequence for the y ions and a shorter run for the b ions. There is some overlap of the b and y ions, however, so we have a complementary set.

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Can’t account for dominant ions!!!

Incorrect IDIncorrect ID

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BUT…BUT…

Despite the many of the factors which seemed to lead to a correct identification, it is wrong. Since this is a good quality spectrum, it would be worth pursuing other interpretation options. For one, the peptide may be modified and it would be worth re-searching the data using a database including modifications such as phosphorylation or glycosylation, among others. Several searches may be required. De novo searching would also be a possible approach if modification searches do not provide acceptable results.

Bad Spectral QualityBad Spectral Quality

Good Spectral QualityGood Spectral Quality

Good ScoreGood Score Bad ScoreBad Score

Bad IDBad ID

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Example 2Example 2

No significant hits to reportUnassigned queries: (no details means no match)  Query   Observed   Mr(expt)   Mr(calc)   Delta  Miss Score  Rank   Peptide   

1     868.07    1734.13    1730.98     3.15    2    29     1    KGVASTDNTLIARSLGK

All All dominant ionsdominant ions are are unidentifiedunidentified, , the the spectrumspectrum is of is of good qualitygood quality, , with good signal to noise and well-separated fragment ionswith good signal to noise and well-separated fragment ions

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Mascot score is below generally accepted thresholds.

# b b++ b* b*++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ #

1 129.18 65.10 112.15 56.58     K             17

2 186.23 93.62 169.20 85.11     G 1603.82 802.41 1586.79 793.90 1585.80 793.40 16

3 285.37 143.19 268.34 134.67     V 1546.76 773.89 1529.73 765.37 1528.75 764.88 15

4 356.45 178.73 339.42 170.21     A 1447.63 724.32 1430.60 715.80 1429.62 715.31 14

5 443.52 222.27 426.49 213.75 425.51 213.26 S 1376.55 688.78 1359.52 680.27 1358.54 679.77 13

6 544.63 272.82 527.60 264.30 526.61 263.81 T 1289.48 645.24 1272.44 636.73 1271.46 636.23 12

7 659.72 330.36 642.69 321.85 641.70 321.36 D 1188.37 594.69 1171.34 586.17 1170.35 585.68 11

8 773.82 387.41 756.79 378.90 755.81 378.41 N 1073.28 537.14 1056.25 528.63 1055.27 528.14 10

9 874.93 437.97 857.90 429.45 856.91 428.96 T 959.18 480.09 942.15 471.58 941.16 471.09 9

10 988.09 494.55 971.06 486.03 970.07 485.54 L 858.07 429.54 841.04 421.02 840.06 420.53 8

11 1101.25 551.13 1084.21 542.61 1083.23 542.12 I 744.91 372.96 727.88 364.45 726.90 363.95 7

12 1172.32 586.67 1155.29 578.15 1154.31 577.66 A 631.75 316.38 614.72 307.87 613.74 307.37 6

13 1328.51 664.76 1311.48 656.24 1310.50 655.75 R 560.67 280.84 543.64 272.33 542.66 271.83 5

14 1415.59 708.30 1398.56 699.78 1397.57 699.29 S 404.49 202.75 387.46 194.23 386.47 193.74 4

15 1528.75 764.88 1511.72 756.36 1510.73 755.87 L 317.41 159.21 300.38 150.69     3

16 1585.80 793.40 1568.77 784.89 1567.79 784.40 G 204.25 102.63 187.22 94.11     2

17             K 147.20 74.10 130.17 65.59     1

There are only short runs of contiguous sequence, there is little complementarity between the b and y ions. With dominant ions unidentified the assignment is obviously incorrect. Since the spectrum is of good quality, the next step should be to consider modifications.

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Bad Spectral QualityBad Spectral Quality

Good Spectral QualityGood Spectral Quality

Good ScoreGood Score Bad ScoreBad Score

Good IDGood ID

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Example 3Example 3

Score = 31 below threshold

# b b0 Seq. y y* y0 #

1 72.09   A       15

2 129.14   G 1409.58 1392.55 1391.57 14

3 186.19   G 1352.53 1335.50 1334.52 13

4 257.27   A 1295.48 1278.45 1277.46 12

5 328.35   A 1224.40 1207.37 1206.38 11

6 427.48   V 1153.32 1136.29 1135.31 10

7 526.61   V 1054.19 1037.16 1036.17 9

8 639.77   I 955.06 938.03 937.04 8

9 740.88 722.86 T 841.90 824.87 823.88 7

10 869.99 851.98 E 740.79 723.76 722.78 6

11 967.11 949.10 P 611.68 594.65 593.66 5

12 1096.23 1078.21 E 514.56 497.53 496.54 4

13 1233.37 1215.35 H 385.44 368.41 367.43 3

14 1334.47 1316.46 T 248.30 231.27 230.29 2

15     K 147.20 130.17   1

All dominant ions accounted forLargest peak corresponds to y5 – cleavage at proline

BUT…

•Chemistry is plausible•A Sequest search provided the same identification as the Mascot search•Likely correct assignment

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No water loss for b ions until b9 when Thr appears.

Water loss for y ions also starts after the threonine.

Possibly the presence of the basic histidine and the acidic glutamic acid inhibit the water loss at y3 and y4.

Bad Spectral QualityBad Spectral Quality

Good Spectral QualityGood Spectral Quality

Good ScoreGood Score Bad ScoreBad Score

Bad IDBad ID

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Example 4Example 4

Score = 18 Very weak spectrumScore = 18 Very weak spectrum

There is no baseline, thus we must assume this is a noise spectrum. The ions are likely assigned by random chance.

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# b b++ b* b*++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ #

1 58.06 29.53         G             25

2 157.19 79.10         V 2413.73 1207.37 2396.70 1198.85 2395.71 1198.36 24

3 228.27 114.64         A 2314.60 1157.80 2297.57 1149.29 2296.58 1148.79 23

4 299.35 150.18         A 2243.52 1122.26 2226.49 1113.75 2225.50 1113.25 22

5 412.51 206.76         L 2172.44 1086.72 2155.41 1078.21 2154.42 1077.72 21

6 513.61 257.31     495.60 248.30 T 2059.28 1030.14 2042.25 1021.63 2041.26 1021.14 20

7 600.69 300.85     582.68 291.84 S 1958.17 979.59 1941.14 971.08 1940.16 970.58 19

8 715.78 358.39     697.77 349.39 D 1871.10 936.05 1854.06 927.54 1853.08 927.04 18

9 812.90 406.95     794.88 397.95 P 1756.01 878.51 1738.98 869.99 1737.99 869.50 17

10 883.98 442.49     865.96 433.48 A 1658.89 829.95 1641.86 821.43 1640.87 820.94 16

11 983.11 492.06     965.09 483.05 V 1587.81 794.41 1570.78 785.89 1569.80 785.40 15

12 1111.24 556.12 1094.21 547.61 1093.23 547.12 Q 1488.68 744.84 1471.65 736.33 1470.66 735.84 14

13 1182.32 591.66 1165.29 583.15 1164.30 582.66 A 1360.55 680.78 1343.52 672.26 1342.53 671.77 13

14 1295.48 648.24 1278.45 639.73 1277.46 639.24 I 1289.47 645.24 1272.44 636.72 1271.45 636.23 12

15 1394.61 697.81 1377.58 689.29 1376.60 688.80 V 1176.31 588.66 1159.28 580.14 1158.29 579.65 11

16 1507.77 754.39 1490.74 745.87 1489.76 745.38 L 1077.18 539.09 1060.15 530.58 1059.16 530.08 10

17 1622.86 811.93 1605.83 803.42 1604.84 802.93 D 964.02 482.51 946.99 474.00 946.00 473.51 9

18 1723.96 862.49 1706.93 853.97 1705.95 853.48 T 848.93 424.97 831.90 416.45 830.91 415.96 8

19 1795.04 898.03 1778.01 889.51 1777.03 889.02 A 747.82 374.42 730.79 365.90 729.81 365.41 7

20 1882.12 941.56 1865.09 933.05 1864.11 932.56 S 676.74 338.88 659.71 330.36 658.73 329.87 6

21 1997.21 999.11 1980.18 990.59 1979.19 990.10 D 589.67 295.34 572.64 286.82 571.65 286.33 5

22 2096.34 1048.68 2079.31 1040.16 2078.33 1039.67 V 474.58 237.79 457.55 229.28 456.56 228.79 4

23 2209.50 1105.26 2192.47 1096.74 2191.49 1096.25 L 375.45 188.23 358.42 179.71 357.43 179.22 3

24 2324.59 1162.80 2307.56 1154.28 2306.58 1153.79 D 262.29 131.65 245.26 123.13 244.27 122.64 2

25             K 147.20 74.10 130.17 65.59     1

Although there are some complementary runs of contiguous sequence ions, it is unlikely they are significant, given the quality of the mass spectrum.

We do see a dominant fragment ion at the y17 proline, however other large signals do not correspond to expected cleavages C-terminal of the acidic amino acids.

The assignment of doubly-charged ions without the presence of a basic ion is highly unlikely, therefore this identification is incorrect.

This spectrum could likely be discarded.NEXT

Bad Spectral QualityBad Spectral Quality

Good Spectral QualityGood Spectral Quality

Good ScoreGood Score Bad ScoreBad Score? ID? ID

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Example 5Example 5

# b b++ b* b*++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ #

1 132.20 66.60         M             9

2 261.32 131.16     243.30 122.15 E 945.06 473.04 928.03 464.52 927.05 464.03 8

3 358.43 179.72     340.42 170.71 P 815.95 408.48 798.92 399.96 797.93 399.47 7

4 472.54 236.77 455.51 228.26 454.52 227.76 N 718.83 359.92 701.80 351.40 700.82 350.91 6

5 559.61 280.31 542.58 271.80 541.60 271.30 S 604.73 302.87 587.70 294.35 586.71 293.86 5

6 672.77 336.89 655.74 328.38 654.76 327.88 L 517.65 259.33 500.62 250.81 499.63 250.32 4

7 828.96 414.98 811.93 406.47 810.95 405.98 R 404.49 202.75 387.46 194.23 386.47 193.74 3

8 930.07 465.54 913.04 457.02 912.05 456.53 T 248.30 124.66 231.27 116.14 230.29 115.65 2

9             K 147.20 74.10 130.17 65.59     1

Score = 38

Most abundant peak results from loss of water from serine, not proline cleavage as expected.The doubly-charged y8 ion is reasonable, given the presence of the arginine. However, the b2 doubly-charged ion is unlikely.

Score slightly below threshold, spectral quality OK but fragmentation is limited, questionable ID

The identification could possibly be disregarded if the protein assignment was confirmed by another peptide.

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Bad Spectral QualityBad Spectral Quality

Good Spectral QualityGood Spectral Quality

Good ScoreGood Score Bad ScoreBad Score

Good IDGood ID

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Example 6Example 6

# b b++ b* b*++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ #

1 132.20 66.60         M             14

2 233.31 117.16     215.29 108.15 T 1689.83 845.42 1672.79 836.90 1671.81 836.41 13

3 348.39 174.70     330.38 165.69 D 1588.72 794.86 1571.69 786.35 1570.71 785.86 12

4 476.52 238.77 459.49 230.25 458.51 229.76 Q 1473.63 737.32 1456.60 728.80 1455.62 728.31 11

5 605.64 303.32 588.61 294.81 587.63 294.32 E 1345.50 673.25 1328.47 664.74 1327.49 664.25 10

6 676.72 338.86 659.69 330.35 658.70 329.86 A 1216.39 608.70 1199.35 600.18 1198.37 599.69 9

7 789.88 395.44 772.85 386.93 771.86 386.44 I 1145.31 573.16 1128.28 564.64 1127.29 564.15 8

8 918.01 459.51 900.98 450.99 899.99 450.50 Q 1032.15 516.58 1015.12 508.06 1014.13 507.57 7

9 1033.10 517.05 1016.07 508.54 1015.08 508.05 D 904.02 452.51 886.99 444.00 886.00 443.50 6

10 1146.26 573.63 1129.23 565.12 1128.24 564.63 L 788.93 394.97 771.90 386.45     5

11 1332.47 666.74 1315.44 658.22 1314.46 657.73 W 675.77 338.39 658.74 329.87     4

12 1460.60 730.80 1443.57 722.29 1442.59 721.80 Q 489.55 245.28 472.52 236.77     3

13 1646.82 823.91 1629.78 815.40 1628.80 814.90 W 361.42 181.22 344.39 172.70     2

14             R 175.21 88.11 158.18 79.59     1

Score = 79Complete b and y seriesPlausible ion chemistryCorrect ID!Correct ID!

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Abundant ions at D and W cleavages

The following examples compare spectra that were acquired on a Q-TOF and an ion trap.

Both correct and incorrect identifications are shown.

The different appearance of the fragmentation spectra, and the presence of immonium ions and low mass fragment ions in the Q-TOF, means that different considerations may need to be taken into account when different instruments are being used.

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QTOF

Ion Trap

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Correct Identification

Note the presence of y1 and the immonium ion for F in the QTOF spectrum. The QTOF spectrum shows b ion suppression except for b2.

QTOF

Ion Trap

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Incorrect Identification

Both spectra have many dominant ions unaccounted for. The dominant ions are assigned as b type in the QTOF spectrum, which is unlikely.

Look at the next two slides.

Can you guess which are the correct identifications and which are incorrect?

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QTOF

Ion Trap

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QTOF

Ion Trap

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When interpreting the results of search engines it is important to – Look at the score– Look at the sequence runs– Consider the ion fragmentation chemistry– What about the instrument?– Does it all make sense?

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Summary