Formation of Ammonium Carboxylate Between Sinapinic Acid and Lysine-Containing Proteins

Supplementary Material

Possible evidence of amide bond formation between sinapinic acid and lysine-containing bacterial proteins bymatrix-assisted laser desorption/ionization (MALDI) at 355 nm

Clifton K. Fagerquist*, Omar Sultan and Michelle Q. Carter

WesternRegionalResearchCenter, Agricultural Research Service,

U. S. Department of Agriculture, 800 Buchanan Street, Albany, California94710

Running title: Amide bond of sinapinic to lysine residues

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*Correspondence to: C. K. Fagerquist, Western Regional Research Center, Agricultural Research Service, U. S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. E-mail: . Mention of a brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture over other of a similar nature not mentioned. This article is a US Government work and is in the public domain in the USA.

Figure captions

Supplemental Figure 1

Simultaneous MS/MS (PSD) of the disulfide-intact (HdeA + adducts) precursor ion at m/z 9958 and m/z 9971 [shown in Figure 1 (b)]analyzed with different TIS window values on the low mass side of the precursor ion at m/z 9958.

Supplemental Figure 2

SimultaneousMS/MS (PSD) of the disulfide-reduced (HdeA + adducts) precursor ions at m/z 9957 andm/z 9971 analyzed with different TIS window values on the low mass side of the precursor ion at m/z 9957.

Supplemental Figure 3

(a) MS spectrum of an un-fractionated cell lysate of strain MQC238 (with HdeB) ionized with CHCA matrix. Expanded mass range is shown as insert. (b) MS spectrum of the same cell lysate ionized with SA matrix. Expanded mass range is shown as insert.

Supplemental Figure 4

MS/MS (PSD) of the disulfide-intact HdeB precursor ion at m/z 9070 shown in Supplemental Figure 3 (b). (C), (d) and insert in (a) are expanded m/z ranges of (a). Fragment ions are identified by their m/z and their corresponding fragment ion type. Two fragment ions shown in the insert in (a) and in (d) are the result of a double cleavage of the polypeptide backbone and are identified by two fragment ion types. A curly bracket in (d) indicate a fragment ion triplet due to polypeptide backbone cleavage between the two cysteine residues and symmetric and asymmetric cleavage of the disulfide bond. The sequence of HdeB is provided in (b). An asterisk in the sequence indicates a site of polypeptide backbone cleavage with the corresponding fragment ion type above and/or below the cleavage site. A red asterisk indicates that the cleavage also contributes to a double cleavage of the polypeptide backbone. A  superscript indicates the fragment ion is part of a fragment ion triplet. The two cysteine residues that form an intramolecular disulfide bond are boxed.

Supplemental Figure 5

Simultaneous MS/MS (PSD) of the disulfide-intact (HdeB + adducts) precursor ions at m/z 9283 and m/z 9295 shown in Supplemental Figure 3 (b). (C) is an expanded m/z range of (a). Fragment ions are identified by their m/z and their corresponding fragment ion type. The additional fragment ions (which also do not appear in Supplemental Figure 4) indicate adduct attachment and are highlighted by their difference in mass (or m/z) to the corresponding fragment ion without an adduct. The HdeB sequence is shown in (b). An asterisk in the sequence indicates a site of polypeptide backbone cleavage with the corresponding fragment ion type above and/or below the cleavage site. A  superscript indicates the fragment ion is part of a fragment ion triplet. The two cysteine residues that form an intramolecular disulfide bond are boxed.

Supplemental Figure 6

Simultaneous MS/MS (PSD) of the disulfide-intact (HdeB + adducts) precursor ion at m/z 9283 and m/z 9295 [shown in Supplemental Figure 3 (b)]analyzed with different TIS window values on the low mass side of the precursor ion at m/z 9283.

Supplemental Figure 7

MS/MS (PSD) of the disulfide-reduced HdeB precursor ion at m/z 9072. (C) and (d) are expanded m/z ranges of (a). Fragment ions are identified by their m/z and their corresponding fragment ion type. The HdeB sequence is shown in (b). An asterisk in the sequence indicates a site of polypeptide backbone cleavage with the corresponding fragment ion type above and/or below the cleavage site. The two cysteine residues are boxed.

Supplemental Figure 8

Simultaneous MS/MS (PSD) of the disulfide-reduced (HdeB + adducts) precursor ions at m/z 9284 and m/z 9296. (C), (d) and insert in (a) are expanded m/z ranges of (a). Fragment ions are identified by their m/z and their corresponding fragment ion type. The additional fragment ions (whichalso do not appear in Supplemental Figure 7) indicate adduct attachment and are highlighted by their difference in mass (or m/z) to the corresponding fragment ion without an adduct. The HdeB sequence is shown (b). An asterisk indicates a site of polypeptide backbone cleavage with the corresponding fragment ion type above and/or below the cleavage site. The two cysteine residues are boxed.

Supplemental Figure 9

Simultaneous MS/MS (PSD) of the disulfide-reduced (HdeB + adducts) precursor ion at m/z 9284 and m/z 9296 analyzed with different TIS window values on the low mass side of the precursor ion at m/z 9284.