Tandem MS Interpretation Workshop

srhart

Download

Post to :

URL :

Related Presentations :

Add to Flag Embed

Views: 260

Category: Science & Tech..

Presentation Description

No description available.

Comments

Presentation Transcript

Slide 1:

Sarah R Hart This version of the presentation should be used to supplement the materials used in class to aid your interpretation skills All materials herein except where acknowledged otherwise are copyright of Dr SR Hart, Keele University School of Medicine Please do not redistribute this material without permission Interpretation of peptide tandem mass spectra – Podcast version

Interpretation of peptide tandem mass spectra :

Interpretation of peptide tandem mass spectra Practical hints & tips on interpretation Worked example of a peptide MS/MS spectrum

Anatomy of an MS/MS spectrum :

Anatomy of an MS/MS spectrum Precursor ion mass Charge state C-terminal residue Lys, Arg y1 ions (Tryptic peptides) N-terminal residue pair b2/a2 ions Immonium ions Signatures ‘Ladder’ fragment ions Neutral loss fragment ions Internal ions

Precursor ion mass and charge state :

Precursor ion mass and charge state Mass selected for MS/MS = 671.3

Precursor ion mass and charge state :

Precursor ion mass and charge state 0.5 Th 0.5 Th 0.5 Th

Precursor ion mass and charge state :

Precursor ion mass and charge state 0.5 Th 0.5 Th 0.5 Th m/z = 671.3/2 M = (m x z) – z M = (671.3 x 2) -2 = 1341.6 [M+H]+ = 1342.6

Anatomy of an MS/MS spectrum :

C-terminal residue assignment :

C-terminal residue assignment m/z 145-180 Region containing unmodified C-terminal residue m/z

C-terminal residue assignment :

C-terminal residue assignment y1 Arginine 175.1

C-terminal residue assignment :

C-terminal residue assignment y1 Lysine 147.1

C-terminal residue assigned :

C-terminal residue assigned y1 R y1

Anatomy of an MS/MS spectrum :

y and b ion formation :

y and b ion formation

b and a ion formation :

b and a ion formation

N-terminal residue assignment :

N-terminal residue assignment Region containing unmodified N-terminal residue pair m/z (more variable than y1)

Table of b2 ion masses :

http://169.230.19.26:8080/prospector/4.27.1/html/misc/dipep.htm Table of b2 ion masses

N-terminal residue assignment :

N-terminal residue assignment D=m/z 28

Potential C-terminal residue pairs assigned :

Potential C-terminal residue pairs assigned y1 R y1 b2 a2 [IL]N N[IL] [QK]V V[QK] RA AR b2

Anatomy of an MS/MS spectrum :

Immonium ion assignment :

Immonium ion assignment Region containing immonium m/z

Immonium ion region :

Immonium ion region

Immonium ions assigned :

Immonium ions assigned y1 R, (V [IL] Y) y1 b2 a2 [IL]N N[IL] [QK]V V[QK] RA AR b2 [IL]Im YIm VIm

Anatomy of an MS/MS spectrum :

‘Ladder’ sequencing :

‘Ladder’ sequencing [M+H]+ = 1342.6 Look at highest m/z product ion

Assignment of highest y ion :

Assignment of highest y ion [M+H]+ = 1342.6 - 1228.5 = 113, [IL]

Working our way down from highest m/z y ion :

Working our way down from highest m/z y ion 1228.5-114.5 = 114, N

Walking the ladder :

Walking the ladder

Walking the ladder :

Walking the ladder

Finishing the ladder :

Finishing the ladder y1 y2

Climbing back up the ladder (b ions) :

Climbing back up the ladder (b ions) b2 b3 a3

Complete product ion sequence assigned :

Complete product ion sequence assigned y1 [IL]NVYFDEATNR y1 b2 a2 b2 [IL]Im YIm y10 y10 y9 y8 y7 y6 y5 y4 y3 y2 y9 y8 y7 y6 y5 y4 b3 b3 y2 y3

Anatomy of an MS/MS spectrum :

Neutral losses :

Neutral losses -17 (NH3)

Neutral losses :

Neutral losses -18 (H2O)

Anatomy of an MS/MS spectrum :

Internal ions :

Internal ions [FDEA-H2O] NVY

Anatomy of an MS/MS spectrum :

Another example :

Another example

Precursor ion mass :

Precursor ion mass 0.5 Th 0.5 Th [M+2H]2+ = 785.8 M = (785.8 x 2) + 2 [M+H]+ = 1570.6

y1 ion m/z :

y1 ion m/z y1

b2 and a2 :

b2 and a2

b2 ion at m/z 187 :

b2 ion at m/z 187

Immonium ions :

Immonium ions VIm NIm EIm FIm

Sequence so far :

Sequence so far EG GE DA AD VS SV R, (NEF) b2 y1

Ladder sequencing :

Ladder sequencing 1571-1285 = 286

Sequence assigned :

Sequence assigned EG GE DA AD VS SV VNDNEEGFFSAR b2 y1 b3 y11 y2 y3 y4 y5 y6 y7 y8 y9 y10 b4

Checklist: Interpretation of MS/MS Spectra :

Checklist: Interpretation of MS/MS Spectra Calculate the mass of the peptide compare mass of theoretical proposed peptide with measured peptide mass Inspect low m/z for the y1 ion m/z (y1) = 147 (K) or 175 (R) for tryptic peptides Inspect low m/z for the b2 ion often identified by b2/a2-ion pair (∆28 Th). m/z of b2 used to identify the yn-2-ion. Inspect low m/z for immonium ions may indicate presence of specific amino acids Inspect high m/z for the yn-1 ion indication of C-terminal amino acid from b2-ion

Slide 54:

Extend y-ion series to lower m/z work from yn-2 ion assigning peaks separated by m/z equivalent to specific amino acids. Assign corresponding b-ions if found. Extend b-ion series to higher m/z work from b2-ion assigning peaks separated by m/z equivalent to specific amino acids. Assign corresponding y-ions if found. Reconcile amino acid content with spectrum data check amino acid content agrees with any immonium ions. Consider charge state of peptide - presence of His/Lys/Arg residues Attempt to identify all ions in the spectrum! look for loss of H2O (∆ 18) and NH3 (∆ 17), loss of SO2 from Metox (∆ 64) multiply charged ions and internal fragments. All major product ions should be identified in good spectra