Activity-Based Protein Profiling: The Serine Hydrolases

• p. 2 Detection of FP-Biotin-Reactive Proteins by SDS/PAGE-Western Blotting

  SuperSignal chemiluminescence reagents (Bio-Rad)

Only the authors can say what is right here.

Judge's note: Rests on outside knowledge: SuperSignal chemiluminescent substrates are a Pierce trade name, not a Bio-Rad one. The page 2 image prints '(Bio-Rad)' after SuperSignal, two lines after 'avidin-horseradish peroxidase conjugate (Bio-Rad, 1:2,000 dilution)'. Pierce is named elsewhere in the paper as a supplier (the biotin amine). This cannot be verified from the files; the authors can say which supplier was used.

Second check: another model agreed

• p. 2 Experimental Procedures, preparation of FP-biotin (7)

  (diethylamino)sulfur trifluoride (0.021 ml, 0.10 mmol, 4.0

Only the authors can say what is right here.

Judge's note: Rests on outside knowledge: DAST molar mass 161.2 g/mol and density about 1.22 g/ml. The page 2 image prints 0.021 ml and 0.10 mmol. With those constants 0.021 ml is about 0.159 mmol, and 0.10 mmol would be about 0.013 ml. The mmol and equiv values agree with each other (0.10/0.025 = 4.0), so the volume is the figure that does not fit. No diluted solution is mentioned. Only the authors can say which figure is right; it has no bearing on the results.

Second check: another model agreed

• p. 2 Experimental Procedures, preparation of FP-biotin (7)

  N-hydroxysuccinimide (0.05 g,

• p. 2 Experimental Procedures, preparation of FP-biotin (7)

  0.25 mmol, 10 equiv

Only the authors can say what is right here.

Judge's note: Rests on outside knowledge: N-hydroxysuccinimide molar mass 115.09 g/mol. The page 2 image prints 0.05 g and 0.25 mmol. 0.05 g is 0.43 mmol; even the rounding range 0.045-0.055 g gives 0.39-0.48 mmol. The mmol and equiv values agree with each other (0.25/0.025 = 10), so the mass is the figure that does not fit. Minor: reagent in excess, no effect on results.

Second check: another model agreed

• p. 2 Experimental Procedures, preparation of 6

  sodium periodate (0.31 g,

• p. 2 Experimental Procedures, preparation of 6

  1.56 mmol, 4.1 equiv

Judge's note: Rests on outside knowledge: NaIO4 molar mass 213.89 g/mol. The page 2 image prints 0.31 g, 1.56 mmol and 4.1 equiv. 0.31 g is 1.45 mmol; the rounding range 0.305-0.315 g gives 1.43-1.47 mmol, short of 1.56. The mmol and equiv values agree with each other (1.56/0.38 = 4.1). The gap is about 7% on an excess reagent, a small inconsistency of no consequence.

Second check: another model agreed

• p. 2 Experimental Procedures, NMR data for 4

  1.80–1.20 (m, 20H); MALDI-FTMS (DHB) m/z 291.2088

• p. 2 Experimental Procedures, NMR data for 5

  1.80–1.20 (m, 20H); MALDI-FTMS (DHB) m/z 285.1589

• p. 2 Experimental Procedures, compound 4

  1.80–1.20 (m, 20H)

Only the authors can say what is right here.

Judge's note: Read on the page 2 image. Compound 4: 1H + 2H + 4H + 2H + 20H = 29, while the formula printed for 4 has 31 H, so the aliphatic multiplet would need to be 22H. Compound 5: 1 + 2 + 2 + 2 + 20 = 27, matching C13H27O3P (27 H including OH). 4 has one more ethyl group than 5 yet carries the same 20H multiplet, which suggests the value for 4 was carried over. Integrals of broad multiplets are approximate, so this is rated minor and left as a query.

Second check: another model agreed

• p. 2 Experimental Procedures, NMR data for 6

  1.80–1.20 (m, 16H)

Only the authors can say what is right here.

Judge's note: Read on the page 2 image. Listed integrals 2 + 2 + 16 = 20. The formula printed for 6 has 25 H; less the two acidic protons leaves 23, so 3 H (the OCH2CH3 methyl, which the assignment 'CH3CH2OP' shows is present) are missing, and the multiplet would be 19H. For comparison the multiplet for 5 does include the methyl. Integrals are approximate and acidic protons are often unobserved, so minor; the authors can say whether 16H should be 19H.

Second check: another model agreed

• p. 4 Results, Molecular Characterization, first paragraph

  eluted from a Q Sepharose column between 300 and 450 mM

• p. 4 Fig. 2 legend, panel B

  Elution fractions 7–10 (300–500 mM NaCl) are shown

• p. 4 Fig. 2 legend, panel B

  Elution fractions 7–10 (300–500 mM NaCl) are shown.

Judge's note: Viewed page 4. Fig. 2B has lanes 7 and 9 without FP-biotin and 7, 8, 9, 10 with it; bands I and II are strong in lanes 9 and 10. Methods (page 2-3) give a linear 0-500 mM NaCl gradient collected as 10 x 2.5-ml fractions, so 50 mM per fraction and fraction 10 would be about 450-500 mM. The text's upper limit of 450 mM and the legend's 500 mM are attached to the same panel and the paper does not reconcile them. The text may be quoting the concentration at the start of each fraction, so either form could be legitimate; kept as a minor consistency point.

Second check: another model agreed

• p. 1 Abbreviations footnote

  dihydroxybenzoic acid; APH, acylpeptide hydrolase.

• p. 1 Abbreviations footnote, first entry

  Abbreviations: APH, acylpeptide hydrolase; FAAH

• p. 1 Abbreviations footnote

  APH, acylpeptide hydrolase; FAAH

• p. 1 Abbreviations footnote

  2,5-dihydroxybenzoic acid; APH, acylpeptide hydrolase.

Judge's note: Read the footnote on the page 1 image. Both entries are printed, first and last in the list, so this is not an extraction duplicate. Cosmetic.

Second check: none (judge only)

• p. 2 Experimental Procedures, preparation of 5

  36.2.%

• p. 2

  36.2.%; most of the remaining mass

Judge's note: The page 2 image shows '36.2.%' with a period before the percent sign, so it is not an extraction artefact. The other yields are whole numbers. 0.10/0.28 from the printed masses is 35.7%; that small difference is within rounding of the masses and is not part of the finding.

Second check: none (judge only)

• p. 2 Reaction of Protein Samples with FP-Biotin

  Reactions conducted for longer times (1 hr)

• p. 2 Detection by SDS/PAGE-Western Blotting

  for either 1 h at 25°C

• p. 2 Reaction of Protein Samples

  Ethanol (7.5 µL) was added

Judge's note: Seen on the page 2 image: capital-L 'µL' once against lower-case 'µl'/'ml' everywhere else, and 'hr' once against 'h' elsewhere. House-style inconsistency only; meaning is unaffected.

Second check: none (judge only)

• p. 3 Results, Design and Synthesis, second paragraph

  N-hydoxysuccini-

Judge's note: The page 3 image prints 'N-hydoxysuccini-mide' across a line break with the 'r' missing. The word is spelled correctly as 'N-hydroxysuccinimide' in the Experimental Procedures on page 2.

Second check: none (judge only)

• p. 6 Reference 34

  Kantor, J. A., Hand. P. H., Eggensperger

• p. 6 Reference 34

  Hand. P. H.

Judge's note: Viewed the crop of the reference list on page 6: the mark after "Hand" is a period, unlike every other surname in the list, which is followed by a comma. Not an extraction artefact.

Second check: none (judge only)

• p. 6 Reference 7

  Kato, G. J (1999)

Judge's note: Viewed the crop of the reference list on page 6: there is no period after "J" before "(1999)", while all other entries have a period after each initial. Visible in the image, so not a text-layer loss.

Second check: none (judge only)

• p. 6 Reference 6

  O’Conner, M. D.

Only the authors can say what is right here.

Judge's note: Rests on outside knowledge: that the second author of J. Leukocyte Biol. 60, 555-562 (1996) is M. D. O'Connor, not O'Conner. The page image shows "Smyth, M. J., O’Conner, M. D. & Trapani, J. A. (1996) J. Leukocyte Biol. 60, 555–562" as quoted. The correct spelling cannot be checked from the files; a look at the cited paper or Crossref/PubMed would settle it.

Second check: none (judge only)