2013-01-18 biological MS · 1/17/2014 · 1/27/2014 1 Biological Mass Spectrometry: Ionization, Calculating Mass, Charge S. Barnes & M. Renfrow-UAB 1/14/12 Matthew Renfrow, PhD 6-4681

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Biological Mass Spectrometry: Ionization, Calculating Mass, Charge

S. Barnes & M. Renfrow-UAB 1/14/12

Matthew Renfrow, PhD

6-4681

[email protected]

IIF IIEIIH

Eukaryotic Class II transcriptionpreinitiation complex (PIC)

IIB

RNAP

+1-10-30

RNA polymerase- RNAPTATA-binding protein- TBP

Transcription factor IIB- TFIIBTFIIE- IIETFIIF- IIFTFIIH- IIH

RNA polymerase

Dephosphorylation of RNA polymerase largest subunit

PP PPP

RNA polymerase

multiplekinases

P

RAP30RAP74

TFIIF

Initiation Elongation

PFcp1

Phosphatase

TFIIF (RAP74) – Fcp1 phosphataseinteracting domain

Nguyen BD, Abbott KL, Potempa K, Kobor MS, Archambault J, Greenblatt J, Legault P, Omichinski JGProc Natl Acad Sci U S A. 2003 May 13;100(10):5688-93

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RNA polymerase

Dephosphorylation of RNA polymerase largest subunit

PP PPP

RNA polymerase

multiplekinases

P

RAP30RAP74

TFIIF

Initiation Elongation

PFcp1

Phosphatase

P PP

Fcp1 is a Phosphoprotein

Nguyen BD, Abbott KL, Potempa K, Kobor MS, Archambault J, Greenblatt J, Legault P, Omichinski JGProc Natl Acad Sci U S A. 2003 May 13;100(10):5688-93

[M + 2H]2+

Where are additions of 1st and 2nd

phosphate groups?

ESI FT-ICR MSNEDEGSSSEADEMAKALEAELNDLM

Fcp1 C-terminal fragment after 4 h incubation with CKII

900 1000 1200 1300 m/z

[M + HPO3

+ 2H]2+

1100 1400 1500700 800

p p g p

Monoisotopic elemental mass

http://www.avantilipids.com/index.php?option=com_content&view=article&id=2248&Itemid=415

Monoisotopic amino acid mass

http://www.abrf.org/researchgroups/massspectrometry/eposters/ms97quiz/residuemasses.html

Protein & peptide calculator

http://prospector.ucsf.edu/prospector/mshome.htm

http://web.expasy.org/peptide_mass/

Unimod

http://www.unimod.org/modifications_list.php

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Lipid Calculator

http://pharmacology.ucdenver.edu/lipidcalc/Default.aspx

Carbohydrate calculator

http://web.expasy.org/glycanmass/



Amino acid residue massesAlanine 71.037 Leucine 113.084

Arginine 156.101 Lysine 128.094

Asparagine 114.043 Methionine 131.040

Aspartic acid 115.027 Phenylalanine 147.068

Cysteine 103.009 Proline 97.053

Glutamic acid 129.043 Serine 87.032

Glutamine 128.058 Threonine 101.048

Glycine 57.021 Tryptophan 186.079Glycine 57.021 Tryptophan 186.079

Histidine 137.059 Tyrosine 163.063

Isoleucine 113.084 Valine 99.068

The m/z value of a peptide [M+H]+ is the sum of the residue masses plus 18.015 for H2O plus 1.008. So, what is it for ISLLD?

113.084 + 87.032 + 113.084 +113.084 + 115.027 + 18.015 + 1.008 = 560.334


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NEDEGSSSEADEMAKALEAELNDLM

NEDEGSSSEADE

4 Glutamates1 Asparagine

MAKALEAELNDLM

2 Methionines3 Alaninesp g

2 Aspartates1 Glycine3 Serines1 Alanine

1 Lysine2 Glutamates1 Aspartate1 Asparagine3 Leucines

13501300 1400 1450 1500

[M+2H]2+

[M+2H]2+

[M+HPO3+2H]2+

0 hr

2 hr

Incubation with CKII

13501300 1400 1450 1500

13501300 1400 1450 1500

[M+2H]2+

[M HPO3 2H]

[M+HPO3+2H]2+

[M+(HPO3)2+2H]2+21 hr

[M + 2H]2+

Where are additions of 1st and 2nd

phosphate groups?

ESI FT-ICR MSNEDEGSSSEADEMAKALEAELNDLM

Fcp1 C-terminal fragment

900 1000 1200 1300 m/z

[M + HPO3

+ 2H]2+

1100 1400 1500700 800

p p g p

N E D E G pS S S E A D E M A K A L E A E L N D L M

[M + HPO3 + 3H]3+

[M+ HPO3 + 3H]2+• &[M+ HPO3 +2H]2+

z13•

~ x 10

~ x 2

+ 80 Da

c222+

c11

ECD10 ms

400 600 1200 m/z1000 1400 1600800 1800 2000 2200

c4c6

z17•

z22•

c7c5

c222+

c10

c9

c14

c13

z14•

c18c15

c16

c18

c8

10 ms

2

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13501300 1400 1450 1500

[M+2H]2+

[M+2H]2+

[M+HPO3+2H]2+

0 hr

2 hr

Incubation with CKII

ESI FT-ICR MS

13501300 1400 1450 1500

13501300 1400 1450 1500

[M+2H]2+

[M HPO3 2H]

[M+HPO3+2H]2+

[M+(HPO3)2+2H]2+21 hr

Positive ion mode ESI MS

ATP + kinase (4h)

[M+2H]2+

ATP + kinase (4h)30 min β-elimination + ethanethiol addition

+ 2S*

1350 1400 m/z

+1 pS

1350 1400 m/z

[M+2H]2+

+ 1S*

+ 3S*

Molloy, M.P., Andrews, P.C. Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry. Anal. Chem. 2001, 73, 22, 5387-5394

30 min[M+2H]2+

1 h[M+2H]2+

Control

[M+2H]2+

+ 1S*

+ 2S*

1350 1400 m/z

4h ATP30 min-elim.

1350 1400 m/z

+ 1S*

+ 2S*

5 h[M+2H]2+

ControlNo kinase or ATP

Beta elimination + ethanethiol addition

time course

Positive ion mode ESI MS

ATP + kinase (4h)

[M+2H]2+

ATP + kinase (4h)30 min β-elimination + ethanethiol addition

+ 2S*

1350 1400 m/z

+1 pS

1350 1400 m/z

[M+2H]2+

+ 1S*

+ 3S*

Molloy, M.P., Andrews, P.C. Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry. Anal. Chem. 2001, 73, 22, 5387-5394

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[M+2S* + 3H]3+

~ x 10

N E D E G S* S S* E A D E M A K A L E A E L N D L M

ECD (10 ms)

IRMPD (4W 100 ms)

c23**2+

c24**2+

[M+2S* + 3H]2+• &[M+2S*+2H]2+

400 600 1200 m/z1000 1400 1600800 1800

c4 c5

c6*

c7*

c8**

c10**

c22**2+

c11**

c23

z14• z15

•z17

•

c3c9

**

c5 (23/1 s/n) c7* (38/1 s/n) c8

**c6*

131 87 1312

N E D E G S* S S* E A D E M A K A L E A E L N D L M

400 450 600 m/z550 650 700500 750 800 850

c3 c4 (c6) (c7**)

VL

VL

CL

CL

VH

VH

C1

C1

C C

C C

Hingeregion

FcFab Ser/Thr

GalNAc

Gal

NeuAc

NeuAc

β1,3

α2,3

α2,6

CH2-Pro-Val-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Cys-

(CHO) CHO CHO CHO (CHO)

225 228 230 232 236

CH1

CHVK HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRL SLHR

Trypsin Pepsin

VPSTPPTPSPSTPPTPSPS-NH2

VPSTPPTPSPSTPPTPSPS-NH2*

~

*

Synthetic IgA1 HR + 1 GalNAcECD FT-ICR MS/MS

*

m/z1300120011001000900800700600

* = GalNAc

c6c8

c8*No c6

*

c10

No c10

Helen Cooper

1/27/2014

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IgA1 hinge region O-glycosylation

m/z12001100 13001000 15001400 1600 1700

neuraminidase treated GalNac

Gal

Gal

GalNAc

GalGal

1450 155015001400 1600 1650 m/z

FT-ICR-MS

quadrupole+ SWIFT

1450 155015001400 1600 1650 m/z

1450 155015001400 1600 1650 m/z

GalNAc

Gal

Gal

GalNAcGal

GalNAc

isolated

IRMPDMS / MS

NHMFL ESI FT-ICR Instrument (9.4 T)

frontoctopole

middleoctopole

quadrupole

transferoctopole

ICRCell

atm 3-10 Torr

ESI

differential pumping

GalNAc

GalGal

1450 155015001400 1600 1650 m/z

FT-ICR-MS

quadrupole+ SWIFT

1450 155015001400 1600 1650 m/z

1450 155015001400 1600 1650 m/z

GalNAc

Gal

Gal

GalNAcGal

GalNAc

isolated

IRMPDMS / MS

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m/z12001100 13001000 15001400 1600 1700

neuraminidase + O-glycanase treatedGalNac


de-glycosylated HR peptide

m/z12001100 13001000 15001400 1600 1700

neuraminidase treated GalNac

Gal

Gal

V T V P C P V P S T P P T P S P S T P P T P S P S C C H P R L

[M + 3H]3+

IRMPD

~ x 5

~ x 5

y132+/ b7

y242+

y282+y27

3+

y293+


300 500 900 m/z1100700 1300 1500

[M + 2H]2+

b3

y3b5

y102+

y142+

y162+

y172+

y212+

y222+

y262+

y272+

y292+

GalNac


= GalNAc-Gal

= GalNAc

V T V P C P V P S T P P T P S P S T P P T P S P S C C H P R L

m/z15001450 15501400 16501600 1700 1750

Gal

Gal

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S. Barnes-UAB 1/12/10S. Barnes & M. Renfrow-UAB 1/12/11

MyoglobinMyoglobin


c18ziptip80bump_tunebyhand_3ms #2-11 RT: 0.06-0.56 AV: 10 NL: 6.45E4T: FTMS + p ESI Full ms [500.00-1500.00]

70

80

90

100

ce

940.1850R=40385

z=?802.0407R=50180

z=34

7791248 10097550

c18ziptip80bump_tunebyhand_3ms #2-11 RT: 0.06-0.56 AV: 10 NL: 3.36E4T: FTMS + p ESI Full ms [500.00-1500.00]

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

nda

nce

779.1834R=53019

z=35779.0677R=47940

z=35

779.2957R=50590

z=35

778.9847R=47212

z=35779.3549R=61305

z=35779.7514R=61040

z=?

779.6134R=44343

z=?

778.9245R=52076

z=35

778.8336R=53839

z=?

780.0625R=77856

z=?

RXR-Ligand binding domain

700 800 900 1000 1100 1200 1300 1400m/z

0

10

20

30

40

50

60

Rel

ativ

e A

bun

dan

c 779.1248R=53062

z=35

1009.7550R=40362

z=?

1090.4523R=36345

z=25

757.5110R=56767

z=36 1185.2789R=43943

z=23

1238.7905R=26032

z=?1362.8720R=17608

z=?

778.8 779.0 779.2 779.4 779.6 779.8 780.0m/z

1000 1100 1200 1300 1400 1500 1600 1700m/z

0

100

%

1361225.144

1148.588

1081.069

1022.515

1312.467

1413.476

1530.979

Deconvolution of oxidized forms of -lactoglobulin

m/z

18300 18400 18500 18600 mass0

100

%

1.39e318362

18345

18329

184261837818393 18458

1848918505

1852218540 18575

Junlong ShaoS. Barnes & M. Renfrow-UAB 1/12/11

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total crude lipids

FT-ICR mass spectra of total crude lipids derived from Mtb∆whiB3 growing in macrophages demonstrate the absence of SL-1. (A) Total crude lipid extracts were prepared from Mtb growing inside macrophages and analyzed in the negative ion mode by FT-ICR MS. We observed the presence of Ac2PI species at m/z 835.5261 and 851.5566, which corresponds to their reported theoretical masses. The dimannose specie esterified to three acyl chains (Ac3PIM2) corresponds to mass m/z 1413.8888 was also detected in all the strains tested. Note that the multiple lipoforms of SL-1 were absent in Mtb∆whiB3. (B) The SL-1 region of FT-ICR mass spectrum showed a complete absence of this class of lipids (~m/z 2300 to 2600) in Mtb∆whiB3 (C) FT-ICR mass spectra of the PDIM region.

Benefit of removing salt from tryptic digest

900 1520 2140 2760 3380 4000Mass (m/z)

0

8477.0

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsi

ty

1929.87

2209.971993.96

1868.972148.00

2897.041804.90

2225.00 Salt Contamination

899.0 1519.2 2139.4 2759.6 3379.8 4000.0

Mass (m/z)

0

2.7E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsi

ty

1804.96

2164.04

1994.97

2148.00

1928.961475.78

2290.101868.94

2706.121439.742383.97

1179.65 2502.28 3024.06

ZipTip (desalting)

Trypsin autolysis peak


XXXXXKXXXXRXXXKXXXXKXXXXKKXXXXXXXXXXXRXXXXXXXKXXXXXXXXXXRXXXXXXKXXXXXXKXXXXXXXXXXXXKXXXXXXXKXXXXXXXXKXXXXXKXXXXXXXKXXXXXKXXXXXXXRXXXXXRXXXKXXXXXXXXXXXKRRXXXXXKXXXXXXXRXXXXXXXXKXXXX

Trypsin protease digest(C-terminal side

of R & K)

Reverse phaseC18 chromatography

Mass Spectrometer

Ionize liquid phase Peptides into

gas phase ions

Separate gaseous ions based on size

Measure mass

Output mass

Protein

XXXRXXXXXXKXXXXXRXXXXXXXKXXXXXXXX

XXXXXKXXXXRXXXK XXXXK

XXXXKK XXXXXXXXXXXRXXXXXXXK

XXXXXXXXXXRXXXXXXK XXXXXXK

XXXXXXXXXXXXKXXXXXXXK XXXXXXXXK

XXXXXKXXXXXXXK XXXXXK

XXXXXXXRXXXXXR XXXKXXXXXXXXXXXKR

Output assvalues

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300mass0

100

%

y101100.61

y9987.52

b3375.21

b2262.12

234.13

y1175.12

y2274.19

y8916.49

parent681.45

y4487.32

y5602.32

y6730.43

b8875.42

y111247.69

y7859.45

b4446.23

Bind in acidic

condition,Elute with

polar solvent

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Database of all human known

translated proteins

Do a Theoreticaltrypsin digest

CompareObserved Mass values

MassSpectra

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300mass0

100

%

y101100.61

y9987.52

b3375.21

b2262.12

234.13

y1175.12

y2274.19

y8916.49

parent681.45

y4487.32

y5602.32

y6730.43

b8875.42

y111247.69

y7859.45

b4446.23

Mass values =RepID=H2B2C_HUMANMPEPAKFAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVLKRVHPDTGIWCKAMGIMNSFLNDIFERIAGEASRLAHYNKRSTITSRRSRRPCACCCPASWPSTPCPRAPRRSPSTPAPSESLPGPGARSLPPSLPPRVAGCFVSKGSFQGHLTTSVKESFLCCQSQLMFLASRLVNFRRAHNTKHR>UniRef100_Q6DRA6 Putative histone H2B type 2-D n=1 Tax=Homo sapiens RepID=H2B2D_HUMANMPEPAKFAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVLKRVHPDTGIWCKAMGIMNSFLNDIFERIAGEASRLAHYNKRSTITSRRSRRPCACCCPASWPSTPCPRAPRRSPSTPAPSESLPGPGARSLPPSLPPRVAGCFVSKGSFQGHLTPLVK>UniRef100_Q8N257 Histone H2B type 3-B n=2 Tax=Euarchontoglires RepID=H2B3B_HUMANMPDPSKSAPAPKKGSKKAVTKAQKKDGKKRKRGRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIASEASRLAHYNKRSTITSREVQTAVRLLLPGEL

HUMAN FASTA DATABASE

861.4 879.4921.5

953.4 974.5 988.5

1000.6 1196.6 1217.6 1228.51232.6 1233.7

1249.6 1249.61344.7 1455.81484.6 1514.8

1582.9 1583.91616.8 1726.71759.9 1775.9

1790.6 1853.9

To Theoretical Mass values861.4 879.4

921.5 953.4 974.5 988.5

1000.6 1196.6 1217.6 1228.51232.6 1233.7

1249.6 1249.61344.7 1455.81484.6 1514.8

1582.9 1583.91616.8 1726.71759.9 1775.9

1790.6 1853.91869.9 2286.22302.2 2317.2

Mass values =Formula mass=

amino acid composition

AKHAVSEGTKAVTKYTSSK>UniRef100_P0C1H6 Histone H2B type F-M n=1 Tax=Homo sapiens RepID=H2BFM_HUMANMKCHAGSLSANLPALKVTGWSRCVASRQPITVGVCGPKRPLWPSSEKPFNFHTLAKLHDIGLGNDFLDMTLKAQQQKQKSHATIPKCPPLPWSPTVTLCSCYLMAAASAMAEASSETTSEEGQSIQEPKEANSTKAQKQKRRGCRGSRRRHANRRGDSFGDSFTPYFPRVLKQVHQGLSLSQEAVSVMDSMIHDILDRIATEAGQLAHYTKRVTITSRDIQMAVRLLLPGKMGKLAEAQGTNAALRTSLCAIWQQRK>UniRef100_P57053 Histone H2B type F-S n=1 Tax=Homo sapiens RepID=H2BFS_HUMANMPEPAKSAPAPKKGSKKAVTKAQKKDGRKRKRSRKESYSVYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLPHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSAK>UniRef100_Q7Z2G1 Histone H2B type W-T n=1 Tax=Homo sapiens RepID=H2BWT_HUMANMLRTEVPRLPRSTTAIVWSCHLMATASAMAGPSSETTSEEQLITQEPKEANSTTSQKQSKQRKRGRHGPRRCHSNCRGDSFATYFRRVLKQVHQGLSLSREAVSVMDSLVHDILDRIATEAGRLARSTKRQTITAWETRMAVRLLLPGQMGKLAESEGTKAVLRTSLYAIQQQRK>UniRef100_Q16695 Histone H3.1t n=1 Tax=Homo sapiens RepID=H31T_HUMANMARTKQTARKSTGGKAPRKQLATKVARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLMREIAQDFKTDLRFQSSAVMALQEACESYLVGLFEDTNLCVIHAKRVTIMPKDIQLARRIRGERA>UniRef100_Q6NXT2 Histone H3.3C n=1 Tax=Homo sapiens RepID=H3C_HUMANMARTKQTARKSTGGKAPRKQLATKAARKSTPSTCGVKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFNTDLRFQSAAVGALQEASEAYLVGLLEDTNLCAIHAKRVTIMPKDIQLARRIRGERA>UniRef100_Q99525 Histone H4-like protein type G n=1 Tax=Homo sapiens RepID=H4G_HUMANMSVRGKAGKGLGKGGAKCHRKVLSDNIQGITKCTIRRLARHGGVKRILGLIYEETRRVFKVFLENVIWYAVTNTEHAKRKTVTAMAVVYVLKRQGRTL>UniRef100_O14929 Histone acetyltransferase type B catalytic subunit n=2 Tax=Homininae RepID=HAT1_HUMANMAGFGAMEKFLVEYKSAVEKKLAEYKCNTNTAIELKLVRFPEDLENDIRTFFPEYTHQLFGDDETAFGYKGLKILLYYIAGSLSTMFRVEYASKVDENFDCVEADDVEGKIRQIIPPGFCTNTNDFLSLLEKEVDFKPFGTLLHTYSVLSPTGGENFTFQIYKADMTCRGFREYHERLQTFLMWFIETASFIDVDDERWHYFLVFEKYNKDGATLFATVGYMTVYNYYVYPDKTRPRVSQMLILTPFQGQGHGAQLLETVHRYYTEFPTVLDITAEDPSKSYVKLRDFVLVKLCQDLPCFSREKLMQGFNEDMAIEAQQKFKINKQHARRVYEILRLLVTDMSDAEQYRSYRLDIKRRLISPYKKKQRDLAKMRKCLRPEELTNQMNQIEISMQHEQLEESFQELVEDYRRVIERLAQE>UniRef100_Q969S8 Histone deacetylase 10 n=1 Tax=Homo sapiens RepID=HDA10_HUMANMGTALVYHEDMTATRLLWDDPECEIERPERLTAALDRLRQRGLEQRCLRLSAREASEEELGLVHSPEYVSLVRETQVLGKEELQALSGQFDAIYFHPSTFHCARLAAGAGLQLVDAVLTGAVQNGLALVRPPGHHGQRAAANGFCVFNNVAIAAAHAKQKHGLHRILVVDWDVHHGQGIQYLFEDDPSVLYFSWHRYEHGRFWPFLRESDADAVGRGQGLGFTVNLPWNQVGMGNADYVAAFLHLLLPLAFEFDPELVLVSAGFDSAIGDPEGQMQATPECFAHLTQLLQVLAGGRVCAVLEGGYHLESLAESVCMTVQTLLGDPAPPLSGPMAPCQSALESIQSARAAQAPHWKSLQQQDVTAVPMSPSSHSPEGRPPPLLPGGPVCKAAASAPSSLLDQPCLCPAPSVRTAVALTTPDITLVLPPDVIQQEASALREETEAWARPHESLAREEALTALGKLLYLLDGMLDGQVNSGIAATPASAAAATLDVAVRRGLSHGAQRLLCVALGQLDRPPDLAHDGRSLWLNIRGKEAAALSMFHVSTPLPVMTGGFLSCILGLVLPLAYGFQPDLVLVALGPGHGLQGPHAALLAAMLRGLAGGRVLALLEENSTPQLAGILARVLNGEAPPSLGPSSVASPEDVQALMYLRGQLEPQWKMLQCHPHLVA>UniRef100_Q969S8-4 Isoform 4 of Histone deacetylase 10 n=1 Tax=Homo sapiens RepID=Q969S8-4MGTALVYHEDMTATRLLWDDPECEIERPERLTAALDRLRQRGLEQRCLRLSAREASEEELGLVHSPEYVSLVRETQVLGKEELQALSGQFDAIYFHPSTFHCARLAAGAGLQLVDAVLTGAVQNGLALVRPPGHHGQRAAANGFCVFNNVAIAAAHAKQKHGLHRILVVDWDVHHGQGIQYLFEDDPSVLYFSWHRYEHGRFWPFLRESDADAVGRGQGLGFTVNLPWNQVGMGNADYVAAFLHLLLPLAFEFDPELVLVSAGFDSAIGDPEGQMQATPECFAHLTQLLQVLAGGRVCAVLEGGYHLESLAESVCMTVQTLLGDPAPPLSGPMAPCQSALESIQSARAAQAPHWKSLQQQDVTAVPMSPSSHSPEGRPPPLLPGGPVCKAAASAPSSLLDQPCLCPAPSVRTAVALTTPDITLVLPPDVIQQEASALREETEAWARPHESLAREEALTALGKLLYLLDGMLDGQVNSGIAATPASAAAATLDVAVRRGLSHGAQRLLCVALGQLDRPPDLAHDGRSLWLNIRGKEAAALSMFHVSTPLPVMTGGFLSCILGLVLPLAYGFQPDLVLVALGPGHGLQGPHAALLAAMLRGLAGGRVLALLEEVSWAGWRCCGVGRGKGPVTASVFAPGPELHTPASRDPGPGAEWRGTS>UniRef100_Q969S8-2 Isoform 2 of Histone deacetylase 10 n=1 Tax=Homo sapiens RepID=Q969S8-2MGTALVYHEDMTATRLLWDDPECEIERPERLTAALDRLRQRGLEQRCLRLSAREASEEELGLVHSPEYVSLVRETQVLGKEELQALSGQFDAIYFHPSTFHCARLAAGAGLQLVDAVLTGAVQNGLALVRPPGHHGQRAAANGFCVFNNVAIAAAHAKQKHGLHRILVVDWDVHHGQGIQYLFEDDPSVLYFSWHRYEHGRFWPFLRESDADAVGRGQGLGFTVNLPWNQVGMGNADYVAAFLHLLLPLAFEGQMQATPECFAHLTQLLQVLAGGRVCAVLEGGYHLESLAESVCMTVQTLLGDPAPPLSGPMAPCQSALESIQSARAAQAPHWKSLQQQDVTAVPMSPSSHSPEGRPPPLLPGGPVCKAAASAPSSLLDQPCLCPAPSVRTAVALTTPDITLVLPPDVIQQEASALREETEAWARPHESLAREEALTALGKLLYLLDGMLDGQVNSGIAATPASAAAATLDVAVRRGLSHGAQRLLCVALGQLDRPPDLAHDGRSLWLNIRGKEAAALSMFHVSTPLPVMTGGFLSCILGLVLPLAYGFQPDLVLVALGPGHGLQGPHAALLAAMLRGLAGGRVLALLEENSTPQLAGILARVLNGEAPPSLGPSSVASPEDVQALMYLRGQLEPQWKMLQCHPHLVA>UniRef100_Q969S8-5 Isoform 5 of Histone deacetylase 10 n=1 Tax=Homo sapiens RepID=Q969S8-5MGTALVYHEDMTATRLLWDDPECEIERPERLTAALDRLRQRGLEQRCLRLSAREASEEELGLVHSPEYVSLVRETQVLGKEELQALSGQFDAIYFHPSTFHCARLAAGAGLQLVDAVLTGAVQNGLALVRPPGHHGQRAAANGFCVFNNVAIAAAHAKQKHGLHRILVVDWDVHHGQGIQYLFEDDPSVLYFSWHRYEHGRFWPFLRESDADAVGRGQGLGFTVNLPWNQVGMGNADYVAAFLHLLLPLAFEGGYHLESLAESVCMTVQTLLGDPAPPLSGPMAPCQSALESIQSARAAQAPHWKSLQQQDVTAVPMSPSSHSPEGRPPPLLPGGPVCKAAASAPSSLLDQPCLCPAPSVRTAVALTTPDITLVLPPDVIQQEASALREETEAWAR>UniRef100_Q96DB2

825.4577

825.9485 177 183 0 (R) AHVETLR(Q)

1 859.4520 859.9602 157 163 0 (K) VQELQDK(L)

1 988.5310 989.1201 112 119 1 (K) DLEEVKQK(V)

1 1014.6306 1015.2485 197 205 1 (R) LTARLEALK(E)

1 1017.5364 1018.1645 142 150 0 (K) VAPLGEEFR(E)

1 1026.5942 1027.2160 164 172 0 (K) LSPLAQELR(D)

1 1040.6099 1041.2430 229 237 0 (K) AKPVLEDLR(Q)

1 1052.5960 1053.2165 175 183 1 (R) ARAHVETLR(Q)

1 1098.5790 1099.2363 1Gln->pyro-Glu

155 163 1 (R)QKVQELQDK(L)

1 1115.6055 1116.2670 155 163 1 (R) QKVQELQDK(L)

1 1173.6474 1174.3475 218 228 1 (K) ASEQLKALGEK(A)

1 1179.6984 1180.4387 1Gln->pyro-Glu

238 248 0 (R)QGLLPVLESLK(V)

1 1196.7249 1197.4694 238 248 0 (R) QGLLPVLESLK(V)

1 1216.6783 1217.4134 249 260 0 (K) VSILAAIDEASK(K)

1 1218.5749 1219.3040 206 217 0 (K) EGGGSLAEYHAK(A)

1 1255.6569 1256.4500 36 46 0 (K) DFATVYVEAIK(D)

1 1256.6634 1257.4406 1Gln->pyro-Glu

140 150 1 (R)QKVAPLGEEFR(E)

1 1260.6008 1261.3879 131 139 0 (K) WHEEVEIYR(Q)

1 1266.6365 1267.4328 120 129 0 (K) VQPYLDEFQK(K)

1869.9 2286.22302.2 2317.2

825.4577

825.9485 177 183 0 (R) AHVETLR(Q)

1 859.4520 859.9602 157 163 0 (K) VQELQDK(L)

1 988.5310 989.1201 112 119 1 (K) DLEEVKQK(V)

1 1014.6306 1015.2485 197 205 1 (R) LTARLEALK(E)

1 1017.5364 1018.1645 142 150 0 (K) VAPLGEEFR(E)

1 1026.5942 1027.2160 164 172 0 (K) LSPLAQELR(D)

1 1040.6099 1041.2430 229 237 0 (K) AKPVLEDLR(Q)

1 1052.5960 1053.2165 175 183 1 (R) ARAHVETLR(Q)

1 1098.5790 1099.2363 1Gln->pyro-Glu


1 1115.6055 1116.2670 155 163 1 (R) QKVQELQDK(L)

1 1173.6474 1174.3475 218 228 1 (K) ASEQLKALGEK(A)

1 1179.6984 1180.4387 1Gln->pyro-Glu


1 1196.7249 1197.4694 238 248 0 (R) QGLLPVLESLK(V)

1 1216.6783 1217.4134 249 260 0 (K) VSILAAIDEASK(K)

1 1218.5749 1219.3040 206 217 0 (K) EGGGSLAEYHAK(A)

1 1255.6569 1256.4500 36 46 0 (K) DFATVYVEAIK(D)

1 1256.6634 1257.4406 1Gln->pyro-Glu


1 1260.6008 1261.3879 131 139 0 (K) WHEEVEIYR(Q)

1 1266.6365 1267.4328 120 129 0 (K) VQPYLDEFQK(K)


translated proteins



MassSpectra

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300mass0

100

%

y101100.61

y9987.52

b3375.21

b2262.12

234.13

y1175.12

y2274.19

y8916.49

parent681.45

y4487.32

y5602.32

y6730.43

b8875.42

y111247.69

y7859.45

b4446.23



861.4 879.4921.5

953.4 974.5 988.5

1000.6 1196.6 1217.6 1228.51232.6 1233.7

1249.6 1249.61344.7 1455.81484.6 1514.8

1582.9 1583.91616.8 1726.71759.9 1775.9

1790.6 1853.9

Number

m/z(mi)

m/z(av)

Modifications Start End MissedCleavages

Sequence

1 825.4577 825.9485 177 183 0 (R) AHVETLR(Q)

1 859.4520 859.9602 157 163 0 (K) VQELQDK(L)

1 988.5310 989.1201 112 119 1 (K) DLEEVKQK(V)

1 1014.6306 1015.2485 197 205 1 (R) LTARLEALK(E)

1 1017.5364 1018.1645 142 150 0 (K) VAPLGEEFR(E)

1 1026.5942 1027.2160 164 172 0 (K) LSPLAQELR(D)

1 1040.6099 1041.2430 229 237 0 (K) AKPVLEDLR(Q)

Theoretical mass values


921.5 953.4 974.5 988.5

1000.6 1196.6 1217.6 1228.51232.6 1233.7

1249.6 1249.61344.7 1455.81484.6 1514.8

1582.9 1583.91616.8 1726.71759.9 1775.9

1790.6 1853.91869.9 2286.22302.2 2317.2




825.4577

825.9485 177 183 0 (R) AHVETLR(Q)

1 859.4520 859.9602 157 163 0 (K) VQELQDK(L)

1 988.5310 989.1201 112 119 1 (K) DLEEVKQK(V)

1 1014.6306 1015.2485 197 205 1 (R) LTARLEALK(E)

1 1017.5364 1018.1645 142 150 0 (K) VAPLGEEFR(E)

1 1026.5942 1027.2160 164 172 0 (K) LSPLAQELR(D)

1 1040.6099 1041.2430 229 237 0 (K) AKPVLEDLR(Q)

1 1052.5960 1053.2165 175 183 1 (R) ARAHVETLR(Q)

1 1098.5790 1099.2363 1Gln->pyro-Glu


1 1115.6055 1116.2670 155 163 1 (R) QKVQELQDK(L)

1 1173.6474 1174.3475 218 228 1 (K) ASEQLKALGEK(A)

1 1179.6984 1180.4387 1Gln->pyro-Glu


1 1196.7249 1197.4694 238 248 0 (R) QGLLPVLESLK(V)

1 1216.6783 1217.4134 249 260 0 (K) VSILAAIDEASK(K)

1 1218.5749 1219.3040 206 217 0 (K) EGGGSLAEYHAK(A)

1 1255.6569 1256.4500 36 46 0 (K) DFATVYVEAIK(D)

1 1256.6634 1257.4406 1Gln->pyro-Glu


1 1260.6008 1261.3879 131 139 0 (K) WHEEVEIYR(Q)

1 1266.6365 1267.4328 120 129 0 (K) VQPYLDEFQK(K)

1869.9 2286.22302.2 2317.2

1 1052.5960 1053.2165 175 183 1 (R) ARAHVETLR(Q)

1 1098.5790 1099.2363 1Gln->pyro-Glu 155 163 1 (R)QKVQELQDK(L)

1 1115.6055 1116.2670 155 163 1 (R) QKVQELQDK(L)

1 1173.6474 1174.3475 218 228 1 (K) ASEQLKALGEK(A)

1 1179.6984 1180.4387 1Gln->pyro-Glu 238 248 0 (R)QGLLPVLESLK(V)

1 1196.7249 1197.4694 238 248 0 (R) QGLLPVLESLK(V)

1 1216.6783 1217.4134 249 260 0 (K) VSILAAIDEASK(K)

1 1218.5749 1219.3040 206 217 0 (K) EGGGSLAEYHAK(A)

1 1255.6569 1256.4500 36 46 0 (K) DFATVYVEAIK(D)

1 1256.6634 1257.4406 1Gln->pyro-Glu 140 150 1 (R)QKVAPLGEEFR(E)


translated proteins



MassSpectra

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300mass0

100

%

y101100.61

y9987.52

b3375.21

b2262.12

234.13

y1175.12

y2274.19

y8916.49

parent681.45

y4487.32

y5602.32

y6730.43

b8875.42

y111247.69

y7859.45

b4446.23



861.4 879.4921.5

953.4 974.5 988.5

1000.6 1196.6 1217.6 1228.51232.6 1233.7

1249.6 1249.61344.7 1455.81484.6 1514.8

1582.9 1583.91616.8 1726.71759.9 1775.9

1790.6 1853.9

Hemoglobin Subunit Beta

Hemoglobin Subunit Alpha

Scored Protein match


921.5 953.4 974.5 988.5

1000.6 1196.6 1217.6 1228.51232.6 1233.7

1249.6 1249.61344.7 1455.81484.6 1514.8

1582.9 1583.91616.8 1726.71759.9 1775.9

1790.6 1853.91869.9 2286.22302.2 2317.2




825.4577

825.9485 177 183 0 (R) AHVETLR(Q)

1 859.4520 859.9602 157 163 0 (K) VQELQDK(L)

1 988.5310 989.1201 112 119 1 (K) DLEEVKQK(V)

1 1014.6306 1015.2485 197 205 1 (R) LTARLEALK(E)

1 1017.5364 1018.1645 142 150 0 (K) VAPLGEEFR(E)

1 1026.5942 1027.2160 164 172 0 (K) LSPLAQELR(D)

1 1040.6099 1041.2430 229 237 0 (K) AKPVLEDLR(Q)

1 1052.5960 1053.2165 175 183 1 (R) ARAHVETLR(Q)

1 1098.5790 1099.2363 1Gln->pyro-Glu


1 1115.6055 1116.2670 155 163 1 (R) QKVQELQDK(L)

1 1173.6474 1174.3475 218 228 1 (K) ASEQLKALGEK(A)

1 1179.6984 1180.4387 1Gln->pyro-Glu


1 1196.7249 1197.4694 238 248 0 (R) QGLLPVLESLK(V)

1 1216.6783 1217.4134 249 260 0 (K) VSILAAIDEASK(K)

1 1218.5749 1219.3040 206 217 0 (K) EGGGSLAEYHAK(A)

1 1255.6569 1256.4500 36 46 0 (K) DFATVYVEAIK(D)

1 1256.6634 1257.4406 1Gln->pyro-Glu


1 1260.6008 1261.3879 131 139 0 (K) WHEEVEIYR(Q)

1 1266.6365 1267.4328 120 129 0 (K) VQPYLDEFQK(K)

1869.9 2286.22302.2 2317.2

825.4577

825.9485 177 183 0 (R) AHVETLR(Q)

1 859.4520 859.9602 157 163 0 (K) VQELQDK(L)

1 988.5310 989.1201 112 119 1 (K) DLEEVKQK(V)

1 1014.6306 1015.2485 197 205 1 (R) LTARLEALK(E)

1 1017.5364 1018.1645 142 150 0 (K) VAPLGEEFR(E)

1 1026.5942 1027.2160 164 172 0 (K) LSPLAQELR(D)

1 1040.6099 1041.2430 229 237 0 (K) AKPVLEDLR(Q)

1 1052.5960 1053.2165 175 183 1 (R) ARAHVETLR(Q)

1 1098.5790 1099.2363 1Gln->pyro-Glu


1 1115.6055 1116.2670 155 163 1 (R) QKVQELQDK(L)

1 1173.6474 1174.3475 218 228 1 (K) ASEQLKALGEK(A)

1 1179.6984 1180.4387 1Gln->pyro-Glu


1 1196.7249 1197.4694 238 248 0 (R) QGLLPVLESLK(V)

1 1216.6783 1217.4134 249 260 0 (K) VSILAAIDEASK(K)

1 1218.5749 1219.3040 206 217 0 (K) EGGGSLAEYHAK(A)

1 1255.6569 1256.4500 36 46 0 (K) DFATVYVEAIK(D)

1 1256.6634 1257.4406 1Gln->pyro-Glu


1 1260.6008 1261.3879 131 139 0 (K) WHEEVEIYR(Q)

1 1266.6365 1267.4328 120 129 0 (K) VQPYLDEFQK(K)

% Protein Coverage from Fetalfrom Fetal Hemoglobin Gel Band

1/27/2014

12

Protein Sequence

Hemoglobin Subunit Beta

Hemoglobin Subunit Alpha

Peptides from Hemoglobin Subunit Beta

% Protein Coverage from Fetalfrom Fetal

Hemoglobin Gel Band

Protein SequencePeroxiredoxin - 2

Peroxiredoxin-1 isoform 5

1/27/2014

13

• Assigning charge to an aqueous peptide at a given pH is based on the

• Ionization state of the side chains that have a proton that can ionize.

• Assigning how many charges will be on a given GAS PHASE peptide ion for mass spectrometry is SIMILAR BUT DIFFERENT.

• The “rule of thumb” is that the number of basic sites (side chains and amino terminus) is an estimate of how many protons willand amino terminus) is an estimate of how many protons will associate “be attached” to a peptide ion and therefore what it’s charge state will be.

• Protons that associate (not a covalent bond) with a peptide in a volatile polar solvent do not go to a fixed spot. They can move around

• PLUS…..the presence of a negatively charged side chain doesn’t necessarily “negate” a charge state……then you are mixing two different “exercises” that are similar and have similar terminology but are two different processes.

Documents

2013-01-18 biological MS · 1/17/2014 · 1/27/2014 1 Biological Mass Spectrometry: Ionization, Calculating Mass, Charge S. Barnes & M. Renfrow-UAB 1/14/12 Matthew Renfrow, PhD 6-4681