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(A) <t>SELDI</t> time-of-flight mass spectrum of unmodified rhTIMP-1 shows a molecular mass of 24,775 Da. (B) Mass spectrum of PEG 5K -TIMP-1 shows a molecular mass range of 46,529 Da –62,101 Da, indicating conjugation of 4–7 mPEG-5K chains; the most abundant species possesses 5 mPEG-5K chains. The smaller peaks in the range of 23,398–28,524 are consistent with doubly charged ions of the PEGylated species bearing 4–6 mPEG chains. (C) Mass spectrum of PEG 20K -TIMP-1 shows a molecular mass range of 45,139 Da –65,476 Da, indicating attachment of 1–2 mPEG-20K chains, with the mono-PEGylated species predominant. Doubly charged ions for the mono- and di-PEGylated species are also present, as is a peak for unmodified rhTIMP-1.
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(A) SELDI time-of-flight mass spectrum of unmodified rhTIMP-1 shows a molecular mass of 24,775 Da. (B) Mass spectrum of PEG 5K -TIMP-1 shows a molecular mass range of 46,529 Da –62,101 Da, indicating conjugation of 4–7 mPEG-5K chains; the most abundant species possesses 5 mPEG-5K chains. The smaller peaks in the range of 23,398–28,524 are consistent with doubly charged ions of the PEGylated species bearing 4–6 mPEG chains. (C) Mass spectrum of PEG 20K -TIMP-1 shows a molecular mass range of 45,139 Da –65,476 Da, indicating attachment of 1–2 mPEG-20K chains, with the mono-PEGylated species predominant. Doubly charged ions for the mono- and di-PEGylated species are also present, as is a peak for unmodified rhTIMP-1.

Journal: PLoS ONE

Article Title: PEGylation Extends Circulation Half-Life While Preserving In Vitro and In Vivo Activity of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)

doi: 10.1371/journal.pone.0050028

Figure Lengend Snippet: (A) SELDI time-of-flight mass spectrum of unmodified rhTIMP-1 shows a molecular mass of 24,775 Da. (B) Mass spectrum of PEG 5K -TIMP-1 shows a molecular mass range of 46,529 Da –62,101 Da, indicating conjugation of 4–7 mPEG-5K chains; the most abundant species possesses 5 mPEG-5K chains. The smaller peaks in the range of 23,398–28,524 are consistent with doubly charged ions of the PEGylated species bearing 4–6 mPEG chains. (C) Mass spectrum of PEG 20K -TIMP-1 shows a molecular mass range of 45,139 Da –65,476 Da, indicating attachment of 1–2 mPEG-20K chains, with the mono-PEGylated species predominant. Doubly charged ions for the mono- and di-PEGylated species are also present, as is a peak for unmodified rhTIMP-1.

Article Snippet: Protein samples at 1 mg/ml were mixed in 1∶4 protein-to-matrix volume with the matrix (saturated sinapinic acid in 50% acetonitrile and 0.1% trifluoroacetic acid), and spotted (1 μL) onto a SELDI ProteinChip Gold Array (A-H Format, Bio-Rad).

Techniques: Conjugation Assay