considerations for sample preparation. protein extraction mechanical grinding detergents other...
TRANSCRIPT
Considerations for Sample Preparation
Protein Extraction
• Mechanical grinding• Detergents• Other buffers• Sonication
• http://www.piercenet.com/browse.cfm?fldID=FA97D803-6953-48E4-A7BD-6947D35FE83B
Considerations for mass spectrometry• Salts and buffers in high concentrations can cause
ion suppression and adduct formation in electrospray mass spectrometry
• Detergents can interfere with reversed phase separations
• Proteases
Inhibitors
• Most cells have endogenous proteases that can indiscriminately cleave proteins once cellular structure is disrupted
• The same applies to many PTMs (phosphatases, deacetylases, etc)
Salts
Medicago with detergentRT: 0.00 - 89.99
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
2.72
51.5450.90 55.20
55.382.92 49.1042.19 46.73 55.64
39.9555.99
38.7737.59
4.86 56.4326.925.02 36.3527.035.23 71.53
57.43 61.2232.21 63.076.02 27.5626.25
22.30 65.546.85 15.59 16.7814.80 71.74 76.142.52 76.73 84.2478.25 86.42
NL:1.08E9Base Peak MS Medicago_Protein1_pos
MS 2 of detergentMedicago_Protein1_pos #10567 RT: 47.66 AV: 1 NL: 5.69E5T: FTMS + c NSI d Full ms2 [email protected] [120.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z
0
5
10
15
20
25
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40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
808.50
799.50
774.15
129.10575.69
745.79527.00 646.73453.28
660.41438.28
358.22903.50246.18 972.64 1082.69338.71 1616.56
Lysis buffer
• Compatible lysis buffer• 8 M Urea, 50 mM Tris pH 8.5, 5 mM CaCl2, 50-100 mM
NaCl, plus inhibitors
Detergents
• Mass Spec compatible:• RapiGest (Waters)• ProteaseMax (Promega)
• For other detergents, you need a clean up step prior to LC-MS
• FASP• Precipitation (acetone, chloroform/methanol)
Desalting
• C18 reversed phase material
