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

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25

30

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45

50

55

60

65

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75

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85

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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 808.50@hcd30.00 [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

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45

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55

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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