Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.
In this report we describe a novel approach to the mass spectrometric analysis of whole proteins from gels. The strategy consists of three components: conventional SDS-PAGE gels, reversible negative staining procedures, and passive elution of proteins from gels followed by mass spectrometric analysis. Protein bands are excised from SDS-PAGE gels, destained, and extracted. For gel loadings > or = 25 pmol of soluble protein, the proteins can be directly extracted into a solution consisting of formic acid/water/2-propanol. The recovered protein is suitable for matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization mass spectrometric analysis. For gel loadings < 25 pmol protein, the mass spectrometric response, using the direct extraction procedure, drops off sharply, an outcome that is attributed to protein recovery losses. To offset the protein losses, the extraction procedure is slightly modified by performing the passive extraction of the gel with a saturated MALDI matrix solution. During the extraction period, the matrix is allowed to crystallize, forming a suspension in solution. Protein that elutes from the gel has a chance to cocrystallize with the matrix that can be retrieved for MALDI-MS analysis. This method of "capturing" eluted protein into matrix crystals is sensitive to 1 pmol of recombinant mouse leptin protein (16 kDa) loaded onto SDS-PAGE gels and can be used for proteins as large as 70 kDa. Our strategy has particular application to the characterization of endogenous forms of mature proteins from SDS-PAGE gels.
Cohen
S L
SL
Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York 10021, USA.
Chait
B T
BT
D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D002417:Cattle; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D020738:Leptin; D051379:Mice; D008970:Molecular Weight; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods* / Q000706:statistics & numerical data; D014168:Transferrin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification
D020738:Leptin; D009211:Myoglobin; D011506:Proteins; D011994:Recombinant Proteins; D012996:Solutions; D014168:Transferrin; D012967:Sodium Dodecyl Sulfate; D002256:Carbonic Anhydrases