*2.1. Materials*

From August to December 2017, the *Threads of Time: Tradition and Change in Indigenous American Textiles* exhibition was on display at the Michael C. Carlos Museum at Emory University. The objects in the exhibition had entered the museum from historical private collections, and lacking archaeological context, they were attributed on the basis of style. As part of the technical study in tandem with this exhibition, we undertook mass spectrometry analyses to answer questions about the colorants. Encouraged by the exhibition curator, Dr. Rebecca Stone, we chose to focus on the secondary colors: green, orange, and purple. The central question was whether these colors were obtained from a single dye source or if they were produced through overdyeing or mixing of dyes in the dye pot, or through dyeing naturally colored yarns. The colors of dyed objects selected for this study were determined by eye, with multiple individuals interpreting and agreeing on the colors as orange, green, or purple. Organic color is fugitive and subject to deterioration (fading, color shift, color loss). Even if the visible color of a dye had deteriorated, evidence of the original dye would be expected to remain detectable through chemical analysis for characteristic compounds. Secondary colors can be achieved by the use of mordants with a single primary dye. In these examples, we would expect only one dye source to be identified. The secondary colors of the objects were judged visually without assuming how the object might have originally appeared. If an object appeared blue, it was not assumed that the yellow had faded from a mixed green, and thus, the object was not sampled for green. In the case of orange, in particular, it is possible that a red dye could fade to appear orange. The color was evaluated throughout the object, confirming that protected areas (seams, insides, backs) did not reveal evidence of color loss or shift before a sample was removed to represent a given color.

The objects, along with the relevant samples and the historic time periods from which they originate, are listed in Table 1. Each sample consisted of anywhere from a few plucked fibers of the color of interest to a snippet of yarn from a frayed edge. Yarn snippets varied in size from a few millimeters to 2 cm or more in cases where the edge was already heavily damaged such that sample was readily separating from the object. The samples were collected at the Carlos Museum conservation laboratory, and the colors were determined by consensus of those selecting and observing the collection process. The original collection of Nazca, Wari, and Chancay samples were collected in collaboration with Dr. Cathy Selvius DeRoo, formerly of the Detroit Institute of Arts. These samples were placed into labeled polypropylene microcentrifuge tubes, or, in the case of the plucked fiber samples, placed between two glass microscope slides and taped closed for transport back to the laboratory at Eastern Michigan University (EMU) for analysis. A separate technical study was later undertaken of another Peruvian textile from the Pacatnamú site in northern Peru, attributed to the Lambayeque culture of the Central Andes. The Lambayeque samples were collected in glass vials and shipped to EMU for analysis. Images of the textiles and samples are provided in Supplementary Figures S1–S10.

For comparison through time, samples of similar primary and secondary colors from the Paracas Necropolis period were also included in this study. These were collected at the Museo Nacional de Arqueología, Antropología, e Historia del Perú (MNAAHP) in Lima, Peru, by Anne Paul in 1985 from loose fibers and yarns closely associated with mummy textiles; no fibers were removed from the textiles themselves. The colors of these samples were determined visually by Paul when the samples were collected, and transcribed and confirmed by Jakes [27], also through visual observation. These samples have been stored in a climate-controlled museum storage environment at The Ohio State University, Columbus, OH, USA, since they were collected. The samples come from a total of four different mummy bundles in order to sample a wide range of colors and are listed in Table 2. Images of these samples are provided in the Supplementary Figures S11–S14.

Reference samples from the Peruvian dyes collection prepared by Kay and Erik Antúnez de Mayolo in the 1970s in collaboration with Max Saltzman were included as comparative materials [28] in the latter stages of the project with an emphasis on the yellow dyes. The materials in this collection were prepared on spun sheep's wool, cotton, and Peruvian alpaca yarns, all of unknown origin, both unmordanted and with alum mordant. This collection was also the basis for the widely cited report on Peruvian dye plants [29]. Previous studies of portions of this collection have been reported elsewhere [9]; additional samples not included in the reference collection attributed to Saltzman stored at UCLA, Los Angeles, CA, USA, have been included here. The samples analyzed here were only the alum-mordanted sheep's wool yarns. Additional reference materials were prepared from commercial sources as necessary, particularly for secondary colors; details can be found elsewhere [30].

The solvents used in the methods described here were acetonitrile (ACN) of HPLC grade (Sigma-Aldrich, St. Louis, MO, USA); deionized water (H2O) from a Barnstead Diamond water polishing system; methanol, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO), all of HPLC grade (VWR Chemicals, Radnor, PA, USA); formic acid (FA) of high purity grade (VWR); and hydrochloric acid (HCl) and Na2EDTA both from laboratory stocks. Trifluoroacetic acid (TFA) of ReagentPlus purity (99%) was obtained from Sigma-Aldrich. Dye colorant reference compounds were obtained commercially from a variety of sources. The xanthopurpurin standard was synthesized by T. Friebe and S. Augustin, EMU Chemistry Department.


**Table 1.** Textile samples from the Michael C. Carlos Museum; images in Supplementary Figures S1–S10.

**Table 2.** Paracas Necropolis samples from Museo Nacional de Arqueología, Antropología, e Historia del Perú (MNAAHP), Lima, Peru, collected in 1985 by A. Paul. Identification numbers refer to a specific mummy bundle, specimen, and subspecimen (bundle-specimen subspecimen) as recorded by Paul. Images of samples are found in Supplementary Figures S11–S14.


*2.2. Methods: Ambient Ionization Mass Spectrometry* 2.2.1. DART-MS

A short section (approximately 1 mm) from the end of each yarn sample was cut off using a clean razor blade on a clean glass microscope slide. The resulting loose fibers were divided roughly in half for analysis, with each clump of fibers collected in the tip of cleaned tweezers. The fibers were then introduced into the gap between the DART ionization source (IonSense, Saugus, MA, USA) and Orifice 1 of the AccuTOF mass spectrometer (JEOL USA, Peabody, MA, USA). The helium DART gas was heated to 350–450 ◦C as needed to obtain good signal-to-noise ratios, and spectra were collected in both positive and negative ion

modes using the default grid voltages in the DART controller software. The "peaks voltage" on the AccuTOF was selected for maximum intensity in the range of interest (150–1000 Da), while the ring lens and Orifice 2 voltages were set to ±5 V, depending on whether the spectra were being collected in positive (+) or negative (−) ion modes. Orifice 1 was set to ±30 V (again corresponding to the ionization mode) to minimize fragmentation. One half of the sample was analyzed directly without any preparation, and the second half was treated with approximately 1 μL of formic acid (88%) prior to introduction into the DART ion source gap. Previous studies have shown that acid treatment yields a stronger signal for most dye colorants during DART-MS analysis [25,31].
