Maldi-tof. Basics on Voyager. Basics on Maldi-Tof Basics sample preparation Resolution Delayed Extraction Guide wire/Beam steering Reflector Instrument tuning Calibration Theory. Sample plate. Extraction grids. Reflector detector. Linear detector. Timed ion selector.
Components of a Mass Spectrometer
Voyager-DE and DE PRO
Applied BiosystemsMALDI-TOF instruments
Ion Source: MALDI
(Matrix Assisted Laser Desorption Ionisation)
Laser flash produces matrix (M) neutrals, positive, negative ions and sample neutrals.
M M*, MH+, (M-H)-
Sample molecules (A) are ionised by gas phase proton transfer
2: Expansion of the ion cloud in the absence of an electric field
3: Field applied. Gradient accelerates slow ions more than fast ones.
4: Slow ions catch up with faster ones at the detector
Delayed Extraction (DE)
1: Laser fired. Formed ions detach from plate in the absence of an electric field
Ions with different mass, same Kinetic Energy
Ions with lighter mass will fly faster and will reach the detector first
Flying times of the ions are proportional to m/z ratios
Mass Filter: TOF
(Time Of Flight)
The electrical field applied within the reflector produces an ion mirror effect directing the ions towards a second detector
Voyager Sample Plates
Sinapinic acid (3,5-Dimethoxy-4-hydroxy cinnamic acid)
2,5-dihydroxybenzoic acid (2,5-DHB)
Peptide (0.1-10 pmol/l)
Protein (0.1-10 pmol/ l)
Oligonucleotide (10-100 pmol/ l)
2,4,6-trihydroxy acetophenone (THAP)
2-(4-hydroxyphenylazo)-benzoic acid (HABA)
3-hydroxypicolinic acid (3-HPA)
10 g/L in 30-50% ACN with 0.1% TFA
10 mg/ml in water or 50% ACN
A = 10 mg/ml DHB in 20% ACN
B = 10 mg/ml 5-methoxysalicylic acid in 50% ACN
Combine A:B (9:1)
5 mg/ml in 50% ACN 0,1% TFA
Immediately after, matrix in solvent
And matrix again
Thin Layer (Nitrocellulose)
NC and matrix solution (dry)
Then sample (dry)
TFA on top, blow off with an air supply; repeat
Thin Layer (Acetone)
First matrix in acetone (dry)
On-plate washing possible
Mass / (peak width at half peak height)
RESULT = Better Mass Accuracy
C12 : 5730.61High Resolution - Too much data?Monoisotopic resolution of Insulin
In compounds with more than 100 carbon atoms the height of the 13C isotope peak exceeds the height of the 12C peak
When ions are accelerated they exhibit a broad energy spread. When forming ions in a weak electric field then applying a high voltage pulse after a time delay, this energy spread can be minimized. A potential gradient is formed in the ionization region by the voltages applied to the sample plate and the variable voltage grid.
Ref: W.C.Wiley and I.H.McLaren Rev.Sci.Instrum 1953,26,1150-1157
Ions must line up at the beginning of the flight tube
This initial focus is refocused by the reflector which can be fine tuned for second order velocity focusing.
Refocusing region - some move farther into reflector, than others
Accuracy, Precision & Resolution
None of the darts are close to the true value (bull’s eye) : the measurements are not accurate. Also, since the darts are not very close to each other, the set of measurements is notprecise either.
Since all of the measurements are close together, they areprecise, but since they are not close to the true value, they are not accurate
The measurements are all close to the true value, so they are accurate. Also, the measurements are all close to each other, so they are precise
21 ppm error
Res = 14200
28 ppm error
Res = 4500
55 ppm error
2) Apply calibration
3) Database search
4) Internally calibrate
using “hits” and resubmit
1) Calibrate on
Rank Digest # # (%) SwissProt Species MW (Da) Protein Name
120060 4/16 (25%) P15992YEAST 23748.5 HEAT SHOCK PROTEIN 26.
234118 3/16 (18%) P08468 YEAST 94523.6 PET111 PROTEIN PRECURSOR.
234931 3/16 (18%) P25301 YEAST 52247.7 DNA REPAIR PROTEIN RAD57.
237093 3/16 (18%) P10664 YEAST 38961.1 60S RIBOSOMAL PROTEIN L2A (RP
237100 3/16 (18%) P49626 YEAST 38931.0 60S RIBOSOMAL PROTEIN L2B (RP2).
254802 3/16 (18%) P38863 YEAST 96825.4 HYPOTHETICAL 96.8 KD PROTEIN
1. 4/16 matches (25%). YEAST. HEAT SHOCK PROTEIN 26. ( 23748.5 Da)
submitted matched ppm start end Peptide Sequence Modifications
1274.6610 1274.6017 46.5200 117 126 (K)DIDIEYHQNK(N)
1461.8579 1461.7953 42.8103 176 189 (K)ADYANGVLTLTVPK(L)
1729.9719 1729.9012 40.8452 160 175 (R)VITLPDYPGVDADNIK(A)
1886.0931 1886.0024 48.1161 159 175 (K)RVITLPDYPGVDADNIK(A)
12 unmatched masses: 888.3 998.6 1139.6 1211.8 1225.8 1288.7 1314.8 1350.7 1537.3 1788.0 1820.0 2041.1
The matched peptides cover 19% (41/213 AA's) of the protein.