File Name : 140609 supplemental figure1.tif

Caption : supplemental fig. 1
ms/ms analysis of peaks identified as methyl-lysine (a), n-trimethyl-phenylalanine (b) and n-trimethyl-tyrosine (c)
identification of any peak by ms/ms analysis in the absence of a standard requires the m/z value, fragmentation pattern, and retention time (rt).  similar rts were found in cases of amino acids and their methylated counterparts confirmed by standard compounds (e.g. n-trimethyl-tryptophan 6.0 min and tryptophan 8.7 min).  a. a peak with an m/z value of 161.128 da in positive ionization mode matched the calculated value for the methyl-lysine positive ion with a hydrogen adduct (161.129 da).  the rt is 20.3 min while that of lysine is 23.3 min.  the ms/ms fragmentation pattern matches the methyl-lysine structure; the 130.086 da fragment corresponds to loss of nh2(ch3) (31.043 da), the 84.080 da fragment to loss of nh2(ch3) (31.043) and co2h2 (46.005 da).  while lysine has two amino-groups, we could not determine which of the amino groups is methylated; thus, we tentatively identified the compound as methyl-lysine.  b. a peak with an m/z value of 208.133 da in positive ionization mode matched the calculated value for the trimethyl-phenylalanine positive ion with a hydrogen adduct (208.133 da).  the rt is 5.3 min while that of phenylalanine is 7.3 min.  the ms/ms fragmentation pattern matches the n-trimethyl-phenylalanine structure; the 149.059 da fragment corresponds to loss of of n(ch3)3 (59.074 da), the 131.049 da fragment to loss of n(ch3)3 (59.074 da) and h2o (18.010 da), and the 103.054 da fragment to loss of n(ch3)3 (59.074 da) and co2h2 (46.005 da).  therefore we tentatively identified the peak as n-trimethyl-phenylalanine.  c. a peak with an m/z value of 224.128 da in positive ionization mode matched the calculated value for the trimethyl-tyrosine positive ion with a hydrogen adduct (224.128 da).  the rt is 8.0 min while that of tyrosine is 10.3 min.  ms/ms fragmentation pattern matches the n-trimethyl-tyrosine structure; the 165.054 da fragment corresponds to loss of n(ch3)3 (59.074 da), the 147.044 da fragment to loss of n(ch3)3 (59.074 da) and h2o (18.010 da).  we thus tentatively identified the peak as n-trimethyl-tyrosine.

File Name : 140609 supplemental figure2.tif

Caption : supplemental fig. 2
quantification of plasma and rbc metabolites and ergothioneine derivative peak areas
blood samples were obtained as described in fig. 2.  a. a scatter plot comparison of plasma and rbc samples #1 and #2, as described in fig. 2a.  85 % of peaks were within a 2-fold change in both plasma and rbc samples.  b. scatter plot comparison of identified compounds in plasma and rbc samples as described in fig. 2b.  in both plasma and rbc samples, approximately 96% of the peaks were within a 2-fold range.  c. multiple peaks are ergothioneine derivatives.  arrows indicate primary peaks.  we were able to identify 19 peaks derived from ergothioneine.

File Name : 140609 supplemental figure3.tif

Caption : supplemental fig. 3
scatter plot comparison of compounds quantified in both rbcs and plasma 
peak areas of each compound identified in rbcs and plasma, plotted in a scatter plot.  each compound is indicated with the number with the compound name in the table inset.  purple number and name represent the compounds enriched in rbc (the ratio rbc:plasma is more than 2-fold).