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Meeting S-HPP Chr 16 La Cristalera , 28-29 August 2012 MRM Group Zaragoza IACS PowerPoint Presentation
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Meeting S-HPP Chr 16 La Cristalera , 28-29 August 2012 MRM Group Zaragoza IACS Irene Orera and Sílvia Barceló-Batllori. Equipment Protein quantitation Digestion MRM Strategy: - MRM transition selection MRM transition detection in samples

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Presentation Transcript
slide1

Meeting S-HPP Chr 16

La Cristalera, 28-29 August 2012

MRM Group Zaragoza IACS

Irene Orera and Sílvia Barceló-Batllori

slide2

Equipment

  • Protein quantitation
  • Digestion
  • MRM Strategy: - MRM transition selection
          • MRM transition detection in samples
          • MRM evaluation, specifity and verification (MSMS)
          • MRM optimization (CE)
          • LOD, LOQ and reproducibility
  • Limitations
slide3

Equipment:

    • 4000QTRAP AB Sciex
    • Tempo-nano MDLC
  • Protein quantitation: µg/µL, n=3
  • Protein digestion:
  • In solution digestion:
  • -200mM DTT 30 min 37ºC
  • -200mM IAA 30 min ambient temperature in dark
  • -Trypsin gold (promega) 0.1 µg/µL in 25 mMamoniumbicarbonate: protein ratio 1:20, digestion o.n. 37ºC.
  • - Stop with formic acid and adjust pH<6
slide4

MRM Strategy:

  • MRM transition selection:
  • - MRM pilot 2.0 : -initial peptide selection: 12 – 35 aa, no Met no Cys, 0 missed cleavage, 10 peptides per protein, +2 and +3 charge
  • -automatic fragment selection of 5 highest intensity of y ions (10 ideally)
  • - SRM Atlas
  • - MRM transition detection in samples:
      • HPLC:
        • TrapcolumnAcclaimPepMap 100 nano 100 um ID x 2 cm
        • ColumnAcclaimPepMap 100 nano 75 um x 15 cm
        • 3ug of protein injected
        • 90 min gradient (mobile phase A: 2% ACN, 0.1% Formic Acid, mobile phase B: 98% ACN, 0.1% Formic Acid
slide5

MRM Strategy:

        • HPLC: 90 min gradient (mobile phase A: 2% ACN, 0.1% Formic Acid, mobile phase B: 98% ACN, 0.1% Formic Acid
  • -MS:
        • Ion source: nano ESI, 2800V, CUR 20psi, GS1 20psi, IHT 150ºC, DP 80V
        • 200 transitions/method, dwell time 50 msec
        • MIDAS: MRM + IDA + 2EPIs
slide6

MRM evaluation, specifity and verification

  • a) manual revision of transitions
      • -coelution of different transitions corresponding to the same peptide
          • -good signal intensity and S/N

Fructose-b aldolase

GILAADESTGSIAK

666.9/907.4 +2/y9

666.9/978.5 +2/y10

666.9/1045.5 +2/y11

RT: 39,9 min

slide7

Fructose-b aldolase

GILAADESTGSIAK

666.9/907.4 +2/y9

666.9/978.5 +2/y10

666.9/1045.5 +2/y11

RT: 39,9 min

slide9

b) proteotypic peptides: Blast http://web.expasy.org/blast/ and NCBI blast without gaps 100% identity

slide11

In progress:

    • - MRM optimization (CE)
    • - peptide/transition verification/validation/identification (MSMS)
    • - LOD, LOQ and reproducibility
slide12

Limitations

    • -Validation and specificity of MRM transitions by MSMS/MIDAS (MRM Initiated Detection and Sequencing) difficult due to sample complexity and low sensitivity of our MS equipment.
    • -In highly complex samples like the ones we are dealing, it is highly unlikely that the peptide corresponding to the transition will be within the most intense ones and it will be fragmented ……
  • Alternatives:
    • - validate MSMS in shot-gun high sensitive equipment (orbis or similars)
    • - SILAC labeling of cell culture, for transition/retention time specifity
    • - aqua-synthetic peptides
slide13

Ebhardt, Sabidó et al, Proteomics2012

  • cell culture SILAC label
  • peptide identification with light synthetic peptides added to heavy endogenous peptides
  • for transition/retention time specifity and transition order and relative intensity, allows to eliminate interfering signals
  • heavy synthetic peptides (aqua) for quantitation