Update publication information. Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is widely used in protein structural analysis. In this study we develop a new class of non-hydrolyzable amine-selective di-ortho-phthalaldehyde (DOPA) cross-linkers, one of which is DOPA2. Cross-linking of proteins with DOPA2 is 60-120 times faster than that with the N-hydroxysuccinimide ester cross-linker DSS. Compared with DSS cross-links, DOPA2 cross-links have a higher degree of agreement with the crystal structures of tested proteins. More importantly, DOPA2 has unique advantages of working at low pH, low temperature, or in the presence of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Using staphylococcal nuclease, bovine serum albumin, and bovine pancreatic ribonuclease A, we demonstrate that DOPA2 cross-linking provides abundant spatial information about the conformations of proteins denatured to varying degrees. Lastly, we show that CXMS with only 10 seconds of DOPA cross-linking uncovers conformational changes ass